Gin  5  5-5 


HISTOLOGICAL   TECHNIQUE 


BY 
B.  F.  KINGSBURY,  Ph.D.,  M.D. 

Professor  of  Histology  and  Embryology 


1915 

CARPENTER    &    COMPANY 
ITHACA,  N.Y. 


NOTE. 

The  methods  in  use  in  the  Department  of  Histology  and  Embryol- 
ogy and  applied  in  the  instruction,  have  been  in  the  past  twice 
gathered  together  and  put  in  printed  form  for  the  convenience  of 
students  and  staff:  "Histological  Methods,"  by  Professor  S.  H. 
Gage;  the  technique  portion  of  "Vertebrate  Histology"  by  Gage 
and  Kingsbury.  In  the  following  presentation  the  latter  of  these  two 
has  been  largely  drawn  on  and  certain  loose  sheets  of  technical  direc- 
tions by  S.  H.  Gage  incorporated.  In  addition  the  writer  is  particu- 
larly indebted  to  The  Microscope,  by  S.  H.  Gage;  The  Microtomists 
Vademecum,  by  A.  B.  Lee,  and  Die  Enzyklopaedie  der  mikrosko- 
pischen  Technik,  together  with  a  large  number  of  original  articles, 
some  of  which  are  included  among  the  list  of  References.  When 
referred  to  in  the  text,  the  reference  is  indicated  by  a  bracketed 
number.  Acknowledgment  is  also  due  to  Instructors  in  the  Depart- 
ment, J.  A.  Badertscher  and  H.  M.  Kingery.  Their  names  in 
parenthesis  indicate  methods  particularly  due  to  them. 

B.  F.  KINGSBURY. 


COPYRIGHT 

CARPENTER  &  Co. 

1915 


CONTENTS. 

Page 

INTRODUCTION 5 

FIXATION 7 

Fixers,  List  of   . 11 

ISOLATION 16 

SECTIONING  &  IMBEDDING 19 

Schema  for  Imbedding 21 

The  Paraffin  Method 22 

The  Celloidin  Method '.  26 

The  Freezing  Method 33 

STAINING 34 

Stains,  List  of 38 

Preparation  for  Staining 46 

Schema  for  Staining 51 

MOUNTING,  (Sealing,  Labeling) 52 

Slides  and  Covers,  Knife 57 

SPECIAL  METHODS 59 

The  Cell 59 

Connective  Tissue 61 

Muscle 66 

The  Nervous  System 68 

The  Blood 79 

Fine  Injection 82 

Silver  Nitrate  Impregnations 83 

Histo-chemical  Methods 84 

REFERENCES 90 

INDEX  93 


INTRODUCTION. 

Very  few  structures  of  the  animal  organism  can  be  adequately 
examined  microscopically  without  being  first  subjected  to  a  prepara- 
tory treatment  involving  in  many  cases  the  employment  of  compli- 
cated methods.  Save  in  the  case  of  the  body  fluids  and  certain 
membranes,  animal  tissues  are  bulky,  more  or  less  opaque,  and 
therefore  unsuited  for  examination  under  the  microscope  which 
requires  surface  or  thin  layers  of  substance.  Examination  is  made 
possible  in  such  cases  in  one  of  two  ways, — the  elements  composing 
the  structure  may  be  separated  from  each  other,  or  thin  slices  may  be 
prepared. 

The  above,  however,  presents  but  the  grosser  aspect  of  the  neces- 
sity of  preparation  of  animal  tissues  for  examination  with  the  micro- 
scope. The  histological  analysis  of  bodily  structure  makes  further 
demands  on  the  refinement  of  methods.  Treatment  with  chemicals 
and  stains  (Fixation  and  Staining)  has  for  its  purpose  not  only  the 
preservation  and  delineation  of  structure,  but  its  identification  by 
means  of  more  or  less  definite  chemical  (physical)  reactions.  The 
goal  from  this  side  of  histological  technique  is  an  analysis  from  the 
chemico-physical  as  well  as  the  morphological  aspect  and  the  inter- 
pretation of  morphology  in  terms  of  physiology.  Increase  in  our 
knowledge  of  the  finer  structure  of  the  body  in  the  past  has  been,  as 
advance  in  the  future  will  be,  accompanied  by  and  dependent  on  the 
application  of  a  more  exact  technique  along  these  lines;  while  for 
those  who  aim  to  do  practical  work  in  histology  and  pathology  a 
mastery  of  the  more  important  methods  is  indispensable. 

Furthermore,  in  working  with  chemically  altered  structure 
there  is  always  the  danger  of  losing  sight  of  the  conditions  existent 
in  the  living  protoplasm.  It  is  well,  therefore,  in  addition  to  study 
structure  in  the  living  or  fresh  state,  as  little  altered  from  the  natural 
as  may  be.  There  is  also  very  desirable  the  acquisition  of  skill  in 
the  application  of  simple  methods  which  require  neither  expensive 
apparatus  nor  expenditure  of  time, — methods  which  while  they 
may  not  advance  knowledge,  serve  often  to  meet  the  needs  of  a 
preliminary  examination  or  rapid  clinical  diagnosis. 

Of  the  multitudinous  methods  employed  in  microscopic  work 
only  those  are  here  given  which  meet  the  requirements  for  a  general 
working  knowledge  in  histology.  In  special  investigations,  it  is 

5 


6 

necessary  to  make  a  study  of  the  particular  technical  needs  of  the 
problem,  and  for  this  it  is  well  to  consult  the  larger  works  on  technique 
of  which  may  be  mentioned  the  Encyclopedia  of  Microscopical 
Technique  [6];  The  Microtomist's  Vade-mecum  by  A.  B.  Lee  [30]; 
Physiological  Histology,  by  Gustav  Mann  [36];  Mallory  &  Wright, 
Pathological  Technique  [35].  The  books  of  Gage,  Hardesty  and  v. 
Kahlden-Gierke  will  also  be  found  valuable  for  consultation. 

General  Histological  Technique  involves  then : 

A.  Examining  fresh,  by  either  B.  or  C.     Advantageous  or  necessary 

when  haste  is  required,  or  in  examining  the  tissue  alive. 

B.  Isolation  or  Dissociation.     Separating  out  the  elements  composing 

a  tissue  by  (a)  teasing  or  (b)  treatment  with  reagents  and 
teasing. 

C.  Cutting  thin  sections  of  the  tissue  or  organ. 

For  C.  are  generally  necessary:     . 
1.     Fixing  the  tissue   (§  §  1 — 34).     Hardening. 
%.     Sectioning  by  one  of  the  following  methods : 

(a)  Free-hand,  without  an  imbedding  mass,  or 

(b)  With  an  imbedding  mass,  as 

(1)  By  the  Paraffin  method  (§  50) ,  or 

(2)  By  the  Celloidin  method  (§  61) ,  or 

(3)  By  the  Freezing  method  (§  73) . 

D.  Staining; — to  outline  and  differentiate  the  structure,  or  pick 

out  definite  chemical  substances. 

E.  Mounting; — for   examination   under   the   microscope   and   per- 

manent preservation. 
In  addition, — numerous 

F.  Special  Methods  and  methods  for  the 

G.  Histo-chemical  Analysis  of  structure  must  be  frequently  applied. 


FIXATION. 

§  1.  Fixation  is  one  of  the  fundamental  processes  in  the  ex- 
amination of  plant  and  animal  tissues.  A  fixer  may  be  defined  as 
a  fluid  (or  gas)  into  which  the  living  or  at  least  very  fresh  tissue  is 
placed  in  order  to  preserve  the  structure  of  its  elements  as  nearly  as 
possible  as  in  life.  Living  tissue  when  allowed  to  die  and  remain 
undisturbed,  gradually  loses  the  structural  features  it  had  in  life 
and  undergoes  disintegration  and  decay.  Fixation  depends  upon 
physico-chemical  processes  wherein  the  chemical  constituents  of  the 
tissue  are  thrown  down  in  situ  by  being  rendered  insoluble  in  some 
form  or  represented  by  substitution  products;  the  whole  being  at- 
tended by  as  little  distortion  as  possible.  It  should  be  appreciated 
that  the  chemical  constituents  of  cell  protoplasm  and  of  the  tissues 
are  numerous  and  diverse  in  their  chemical  and  physical  properties, 
so  that  a  universal  or  ideal  fixer  not  only  does  not  exist  but  is  logically 
inconceivable.  The  bulk  of  protoplasm  and  the  tissues  is  protein 
and  the  basis  of  fixation  in  general  is  the  precipitation  or  coagulation 
of  these  chemical  substances.  It  should  be  remembered  however 
that  fats  (lipoids*)  are  a  constant  though  variable  component  of 
cytoplasm;  that  carbohydrates  (glucose,  glycogen,  etc.)  are  usually 
present  in  small  amounts,  and  that  the  products  of  cell  activity  such 
as  secretion-granules,  zymogen,  etc.,  may  be  quite  distinctive  in 
their  physico-chemical  properties.  Within  the  cell  a  certain  "antag- 
onism" exists  between  nucleus  and  cytoplasm,  the  former  oxidative, 
the  latter  reducing,  requiring  often  somewhat  different  fixation 
conditions. 

For  the  best  results,  the  fixer  should  be  chosen  with  a  view  to 
the  preservation  of  some  particular  part  or  constituent,  though  a 
number  of  general  fixers  are  very  serviceable  for  routine  work. 
Rational  fixation  will  depend  upon  a  detailed  knowledge  of  the 
chemical  and  physical  properties  of  the  constituents  that  it  is  de- 
sired to  preserve  and  their  interaction  with  the  chemicals  of  the 
fixer.  In  many  respects,  rational  fixation  still  awaits  further  knowl- 
edge of  the  physics  and  chemistry  of  fixation. 

§  2.  The  chemicals  of  most  service  as  fixers  are:  (1)  osmic 
acid  (osmium  tetroxid),  (2)  platinic  chlorid,  (3)  picric  acid  (tri- 


*Lipoid,  while  not  a  good  chemical  term  is  one  that  is  quite  useful  in  histology, 
to  include  fats,  fatty  acids,  phosphatids,  cholesterol,  etc.,  substances  that  have 
the  same  solvents  and  which  are  found  associated  in  protoplasm. 


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system  (§  §  199,  201,  204)  are;    (IQ)  Muller's  fluid,  (20)  Erlicki's  fluid 
(21)  Potassium  dichromate  (in  aqueous  solution). 

In  the  combination  of  chemicals  in  fixing  solutions  such  as  those 
mentioned  above,  they  should  be  chosen  to  supplement  each  other's 
actions  as  far  a>  po>>ible  and  correct  or  counteract  their  defects.  The 
combinations  must  be  chosen  always  with  a  view  to  the  result  desired 
and  frequently  the  components  and  their  relative  amounts  determined 
empirically, — by  experiment . 

§  7.  The  following  general  rules  should  be  regarded  in  the 
fixation  of  tissues  niid  organs: 

(1).  The  volume  of  the  fixing  fluid  used  should  be  large,  ex- 
ceeding  the  volume  of  the  tissue  at  least  thirty  times.  The  less 
energetic  the  action  of  the  fixer  the  greater  the  amount  of  fluid  to  be 
employed.  When  the  fluid  becomes  turbid  it  should  be  changed  to 
fresh  at  once. 

(2).  Fix  only  as  small  pieces  of  tissue  as  possible,  or  as  is  prac- 
ticable from  the  results  desired.  The  block  of  tissue  should  not 
be  more  than  1  cm.  in  one  dimension,  and  if  possible  let  it  be  much 
shorter, — only  2  to  ,5  mm.  In  some  cases  (Flemming's  and  Hermann's 
fluids)  much  smaller  masses  are  needed  (1  to  2  mm.  thick).  This  is 
desirable  for  the  rapid  and  complete  penetration  of  the  fixer.  Of 
course,  in  the  case  of  entire  organs  it  may  not  be  possible  to  comply 
with  the  conditions.  Fixation  by  injection  may  then  be  resorted  to. 
Physiological  salt  solution  (§  35)  is  first  injected  through  artery  of  the 
organ  or  part  in  order  to  remove  the  blood,  and  this  is  followed  by 
the  injection  of  the  fixing  fluid. 

In  addition  to  these  two  general  principles,  there  are  four  points 
to  be  carefully  considered,  upon  which  the  excellence  in  the  results 
attained  depends;  they  are  (a)  the  fixer  chosen,  (b)  the  time  of 
fixation,  (c)  the  trashing  out  of  the  fixer,  (d)  the  hardening  in  alcohol 
and  the  subsequent  treatment. 

(a)  The  choice  of  a  fluid  into  which  the  tissue  is  placed  should 
be  made  dependent  on  (1)  a  consideration  of  the  particular  feature 
whose  preservation  is  desired  and  the  degree  of  excellence  of  fixation 
that  is  desired  or  necessary,— whether  detail  of  cell  structure  or  the 
structure  of  the  tissue  in  terms  of  cells  or  structural  elements  be 
sought ;  ("2 )  the  penetrating  power  and  the  size  of  the  piece  that  it  is 


10 

necessary  to  have;    and  (3)  the  stain  that  is  desired  subsequently 
which  is  largely  determined  by  the  fixation.* 

(b)  The  time  a  fixer  is  allowed  to  act  should  be  considered  in 
connection  with  the  character  of  the  fluid  and  the  tissue.     Usually 
the  exact  limitation  of  time  is  a  matter  of  secondary  importance 
and  the  tissue  may  remain  in  the  fixer  indefinitely.     In  some  cases, 
however,  its  disregard  affects  the  results  seriously  and  as  a  general 
rule  there  is  a  minimum  and  a  maximum  time  and  between  them 
an  optimum  time  that  should  be  adhered  to. 

(c)  After  the  tissue  has  been  in  the  fixing  fluid  a  proper  length 
of  time,  it  is  necessary  that  it  be  washed  thoroughly  to  remove 
the  fixer  from  it.       Usually  this  should  be  done  by  means  of  water 
or  alcohol  or  both.     In  general,  fixers  containing  salts  insoluble  in 
alcohol  or  but  slightly  soluble,  as  osmic  acid,  chromic  acid,  potassium 
dichromate,  etc.,  should  be  thoroughly  washed  in  water.     Fixers 
containing  picric  acid   or  alcohol   should   always   be   removed   by 
alcohol;    mercuric  chlorid  may  be  washed  out  by  either  water  or 
alcohol. 

Inadequate  washing  out  of  the  fixer  may  either  seriously  affect 
the  cutting  quality  of  the  tissue  (if  it  is  to  be  subsequently  im- 
bedded), the  ease  with  which  it  can  be  stained,  or  there  may  be 
formed  precipitates  in  the  tissue  giving  illusory  effects,  distortions, 
or  at  least  a  dirty  appearance  to  the  preparation.  Time  in  properly 
washing  out  a  fixer  is  always  well  spent,  as  it  is  a  matter  for  serious 
attention. 

§  8.  Resume.  In  brief,  "then:  In  fixing,  take  relatively  large 
amounts  of  fluid  and  small  pieces  of  tissue,  choose  the  fixer  well  with 
a  view  to  the  tissue  and  the  results  desired,  permit  the  fixing  to 
proceed  for  a  sufficient  length  of  time,  and  wash  out  thoroughly. 

HARDENING   AND    STORING. 

§  9.  Each  fixer  has  also  more  or  less  of  a  hardening  action  upon  the  tissue. 
Some  fluids  spoken  of  above  as  fixers  were  primarily  used  as  hardeners,  such  as 
Muller's  fluid  or  Erlicki's  fluid,  while  with  others,  e.  g.,  picric  acid  in  aqueous 
solution,  the  hardening  action  is  a  minimum.  The  hardening  action  of  the  fixer 


*Numerous  papers  have  been  written  on  the  nature  of  fixation  and  the  action 
and  relative  value  of  the  various  chemicals  used  for  that  purpose.  The  contribu- 
tions of  Berg  [4],  Fischer  [7],  Mann  [36],  and  v.  Telly esniczky  [6]  may  be 
particularly  mentioned. 


11 

is  generally  supplemented  by  the  subsequent  use  of  alcohols  of  increasing  strengths 
(50%  to  absolute, — 99%),  as  well  as  in  preparation  for  the  paraffin  and  celloidin 
methods  of  imbedding.  In  fact,  with  modern  methods  of  imbedding  excessive 
hardening  of  the  tissue  is  not  necessary  and  indeed  often  should  be  avoided  as 
affecting  the  cutting  quality  of  the  tissue.  Tissue  after  fixation  has  been  com- 
pleted may  be  stored  in  82  or  95%  alcohol,  or  (better)  imbedded  at  once  (§  47). 

Alcohols.  50%,  67%,  and  82%  alcohols  form  a  series  of  increasing  strengths 
sufficient  for  most  purposes.  They  may  be  prepared  from  95%  alcohol  by  taking 
—(a)  for  50%  alcohol;  95%  alcohol  1  part,  water  1  part;  (b)  for  67%  alcohol; 
95%  alcohol  2  parts,  water  1  part;  (c)  for  82%  alcohol,  95%  alcohol  5  parts, 
water  1  part.  Dilutions  of  other  strengths  may  easily  be  prepared  as  desired 
from  95%  alcohol.  95%  (94%)  alcohol  and  absolute  alcohol  are  necessary  in 
imbedding  by  the  paraffin  and  celloidin  methods  (§  49-). 

§  10.  Stock  Solutions.  It  is  advantageous  to  have  on  hand  strong  solutions 
of  the  chemicals  employed  as  fixers  and  stains.  Where  feasible,  10%  solutions 
are  most  c6nvenient.  The  following  are  the  more  important:  In  aqueous 
solution; — 10%  potassium  dichromate,  10%  copper  dichromate,  10%  chromic 
acid,  10%  platinic  chlorid,  40%  formaldehyde  (formalin),  4%  sodium  sulphate, 
4%  copper  sulphate,  2%  osmic  acid,  saturated  solution  of  mercuric  chlorid, 
saturated  solution  of  picric  acid,  95%  alcohol,  absolute  alcohol,  etc.,  as  well  as 
the  strong  acids,  stock  staining  solutions,  etc. 

FIXERS 
i 

§  11.  Mercuric  chlorid.  One  may  employ  (a)  a  saturated  solution  in 
water  or  (b)  a  saturated  solution  in  normal  salt  solution,  with  1  to  5%  glacial 
acetic  acid.  Water  will  dissolve  about  5%,  normal  salt  solution  about  12% 
of  the  mercuric  chlorid.  This  is  a  good  fixer,  especially  when  the  piece  is  small. 
It  fixes  as  soon  as  it  penetrates  and  is  apt  to  make  tissue  brittle  if  it  is  left  too 
long.  Staining  after  it  is  brilliant.  The  larger  percentage  of  acetic  acid  is, 
perhaps,  to  be  preferred  for  most  histological  objects. 

Fix  the  fresh  tissue  l/2  to  24  hours  according  to  the  size  of  the  piece.  Remove 
to  67%  alcohol  for  1  or  2  days,  82%  alcohol  several  days,  changing  often.  The 
82%  alcohol  should  contain  enough  tincture  of  iodin  to  give  it  a  yellow  color, 
and  fresh  tincture  added  or  the  alcohol  changed  when  the  yellow  color  of  the  iodin 
in  the  alcohol  is  lost.  As  long  as  the  alcohol  is  decolorized,  washing  should  be 
continued,  since  it  is  important  that  the  mercuric  chlorid  be  all  removed  from  the 
tissue;  otherwise  precipitates  will  form  in  the  preparation  after  it  is  mounted 
or  before,  and  spoil  the  result.  Wash  out  in  alcohol  thoroughly  and  carefully. 
Almost  any  stain  may  be  employed  after  a  mercuric  chlorid  fixation. 

§  12.  Zenker's  fluid.  Formula:  Potassium  dichromate,  2.5  grms. ;  sodium 
sulphate,  1  gram;  mercuric  chlorid,  5  grms.;  water,  100  c.c.;  and  add  before 
using,  glacial  acetic  acid,  5  c.  c.  A  stock  solution,  without  the  acetic  acid  should 
be  kept  on  hand.  This  is  a  well  balanced  fixer;  the  potassium  dichromate  seems 
to  check  the  brittleness  that  the  mercuric  chlorid  would  cause  and  improves  the 
fixation  of  the  cytoplasm  while  the  mercuric  chlorid  and  acetic  afford  a  good 
nuclear  fixation.  It  is  distinctly  better  than  mercuric  chlorid;  staining  after  it, 
however,  is  apt  to  be  a  little  more  difficult  and  not  as  brilliant  as  with  mercuric 


12 

chlorid  fixation.  Its  penetration  is  surprisingly  good.  The  sodium  sulphate  is 
probably  unnecessary.  Use  of  physiological  (0.6%)  salt  solution  instead  of  water 
to  prevent  the  formation  of  precipitates  or  the  addition  of  a  small  amount  of  salt 
to  the  fluid  when  made  up  for  use,  is  recommended  by  some. 

Fix  in  Zenker's  fluid  12  to  48  hours,  wash  well  in  water,  running  or  frequently 
changed,  12  to  48  hours,  to  remove  the  dichromate;  transfer  to  67%  alcohol 
for  1  or  2  days,  82%  alcohol  for  several  days,  keeping  in  the  dark  while  in  the 
alcohol.  To  the  82%  alcohol  add  a  drop  or  so  of  tincture  of  iodin,  adding  fresh 
iodin  or  changing  the  alcohol  when  the  color  is  lost.  This  should  be  continued  as 
long  as  the  iodinized  alcohol  is  decolorized  in  order  to  avoid  a  precipitate  of  the 
mercuric  chlorid  in  the  tissue.  Avoid,  however,  adding  an  excess  of  iodin,  since 
it  will  affect  the  staining  quality  of  the  tissue.  Stain  as  you  wish. 

§  13.  Kelly's  fluid.  (Zenker-formol).  Formula:  Zenker's  fluid,  with 
formalin  substituted  for  the  acetic  acid.  Potassium  dichromate,  2.5  grams; 
sodium  sulphate,  1  gram;  mercuric  chlorid,  5  grams;  formalin,  5  or  10  c.  c.,  to  be 
added  before  using.  Valuable  for  the  fixation  of  cytoplasm  and  cytoplasmic 
granules,  where  the  acetic  acid  is  to  be  avoided.  Make  up  from  the  Zenker's 
stock  by  adding  the  formalin. 

Fix  6  to  24  hours  or  longer,  wash  in  running  water  6  to  12  hours,  alcohols. 
If  it  is  desired  to  give  a  longer  mordantage  in  dichromate,  transfer  to  Muller's 
fluid  or  other  simple  dichromate  solution. 

§  14.  Dichromate-acetic.  (Tellyesnicky's  fluid)  Potassium  dichromate, 
3  grms. ;  glacial  acetic, 5 c.  c.;  water,  100  c.  c.  This  fluid  gives  good  preservation 
of  nuclei  and  of  the  cytoplasm,  the  acetic  acid  checking  the  bad  effects  of  the 
dichromate.  A  simpler  fixer  than  Zenker's  fluid. 

Fix  1  to  14  days  according  to  the  size  of  piece  and  the  object.  Wash  in 
running  water  12  or  24  hours,  and  pass  up  through  25,  50,  67,  and  82%  alcohols, 
12  to  24  hours  in  each. 

§  15.  Formol-Dichromate  (Orth's  fluid).  Formula:  Potassium  dichro- 
mate, 2.5  grms. ;  sodium  sulphate,  1  grm.;  water,  100  c.  c.  (i.  e.,  Muller's  fluid); 
formalin,  10  c.  c.  A  good  fixer  of  cytoplasm,  but  an  indifferent  one  for  nuclear 
detail.  It  has  been  especially  used  in  the  case  of  the  nervous  system  (§  199). 
Other  proportions  of  formalin  and  dichromate  may  be  taken,  the  sodium  sulphate 
being  omitted.  It  should  be  freshly  made  up  as  it  soon  deteriorates. 

Fix  3  to  24  hours  or  longer,  wash  in  running  water  12  to  24  hours;  50,  67, 
82%  alcohols,  1  day  in  each.  If  a  long  fixation  is  given,  the  fluid  should  be 
frequently  changed. 

§  16.  Copper  dichromate-sublimate-acetic.  Formula:  10%  copper  dichro- 
mate, 1  part;  4%  copper  sulphate,  1  part;  sat.  solution  mercuric  chlorid,  2 
parts;  glacial  acetic  acid,  rV  to  5%,  according  to  need.  Similar  to  Zenker's 
fluid  in  its  general  properties  and  should  be  used  in  the  same  way.  Excellent  as  a 
preserver  of  cytoplasmic  granules  of  lipoid  nature  (§  177-9).  A  further  mordan- 
tage in  2.5%  copper  dichromate  is  often  advantageous  (3  to  4  days). 

By  the  substitution  of  formalin  (5  to  10%)  for  the  acetic  acid,  a  fixer  compar- 
able to  Kelly's  fluid  (§  13)  is  obtained. 

§  17.  Picro-aceto-formol.  (Picro-formol).  Formula:  Picric  acid,  satu- 
rated aqueous  solution,  75  c.  c.;  formalin,  25  c.  c.;  glacial  acetic  acid,  4  c.  c. 


13 

A  delicate  fixer  useful  in  some  cell  work  and  with  small  objects.  The  formalin 
may  be  omitted  (Picro-acetic). 

Fix  3  to  24  hours,  transfer  to  67%  alcohol,  1  day,  82%  alcohol  several  days 
changing  the  fluid  frequently  as  it  becomes  yellow.  Leave  in  the  alcohol  until 
the  picric  acid  has  been  well  washed  out. 

§  18.  Hermann's  fluid.  Formula:  1%  aq.  sol.  platinic  chlorid,  15  parts; 
2%  aq.  sol.  osmic  acid,  4  parts;  glacial  acetic  acid,  1  part;  or  you  may  take 
10%%aq.  sol.  platinic  chlorid,  3  parts;  1%  aq.  sol.  osmic  acid,  16  parts;  glacial 
acetic  acid,  2  parts;  water,  19  parts.  This  is  generally  recognized  as  one  of  the 
finest  fixers  known,  and  it  is  also  the  most  expensive.  The  form  and  structure 
of  cells  are  well  preserved.  It  should  only  be  employed,  however,  with  very 
small  pieces  of  tissue,  and  is  to  be  used  especially  when  cell  structure  is  to  be 
studied.  Fat  and  the  myelin  of  nerve  fibers  are  stained  black. 

Fix  in  this  1  to  24  hours  (or  longer — days  or  weeks  are  used  by  some),  wash 
well  in  water  (running  or  frequently  changed)  2  to  24  hours,  and  then  place 
in  67%  and  82%  alcohols,  12  to  24  hours  in  each.  In  using  this  fluid,  the  smaller 
the  pieces  taken  the  better  the  fixation  will  be,  and  in  order  that  it  be  possible 
to  obtain  a  good  stain  afterwards  tissue  should  not  be  over-fixed  and  the  fixer 
should  be  thoroughly  washed  out.  If  there  is  a  blackening  of  the  tissue,  or  a 
precipitate  in  it,  both  may  be  removed  by  treatment  of  the  sections  on  the  slide 
with  a  10  or  20%  solution  of  hydrogen  dioxid  in  67%  alcohol,  or  with  perhydrol 
(Merck).  Employ  after  Hermann's  fluid,  as  stains,  Heidenhain's  iron  hema- 
toxylin,  Delafield's  hematoxylin,  safranin  (as  a  red  stain),  or  gentian  violet  (as  a 
blue  stain),  or  neutral  stains. 

§  19.  Flemming's  fluid  (Chrome-aceto-osmic) .  Formula:  1%  aq.  sol. 
chromic  acid,  15  parts;  2%  aq.  sol.  osmic  acid,  4  parts;  glacial  acetic  acid, 
1  part;  or,  10%  aq.  sol.  chromic  acid,  3  parts;  1%  osmic  acid,  16  parts;  glacial 
acetic  acid  2  parts;  water,  19  parts.  This  is  a  fine  fixer  and  in  most  cases  gives 
as  good  results  as  Hermann's,  and  is  not  as  expensive.  It  browns  tissue  less, 
and  while  it  blackens  fat,  does  not  blacken  the  myelin  of  myelinic  nerve  fibers 
as  does  Hermann's.  It  should  be  employed  in  general  in  the  same  cases  and  in 
the  same  way  as  Hermann's  fluid,  and  is  especially  useful  in  the  preservation  of 
free  fats  and  lipoids  (§  227) .  The  presence  of  chromic  acid  gives  it  a  distinct  value 
as  a  cytoplasmic  fixer. 

Fix  tissue  1  to  24  hours  (or  longer);  wash  well  in  running  water  2  to  24 
hours;  place  in  67%  and  82%  alcohols,  12  to  24  hours  in  each.  Bleaching  of 
the  sections  may  be  necessary,  as  with  Hermann's  fluid.  Take  only  very  small 
pieces  of  tissue.  Employ  the  same  stains  as  with  Hermann's  fluid. 

The  reduction  of  the  acetic  acid  to  3  or  4  drops  (about  TJT%)  is  advisable  when 
cytoplasmic  granules  of  lipoid  composition  (§  178)  are  to  be  preserved.  The  fluid 
may  then  be  spoken  of  as  Benda's  fluid. 

§  20.  Mercuro-nitric  (Gilson's  fluid,  modified).  Formula:  Nitric  acid  (46°, 
sp.  gr.  1.456,  80%),  15  c.  c.;  Glacial  acetic  acid,  4  c.  c.;  Mercuric  chlorid,  20  grms.; 
95%  alcohol,  60  c.  c.;  distilled  water,  920  c.  c.  A  good  fixer,  especially  useful 
where  rapid  penetration  is  a  factor,  and  as  a  fixer  of  cells  rich  in  yolk  (amphibian 
ova,  etc.). 

Fix  12  to  24  hours,  transfer  to  50,  67,  and  82%  alcohols.  lodin  should  be 
used  to  ensure  the  removal  of  all  the  mercuric  chlorid  (see  §  11). 


14 

§  21.  Picro-nitric.  Formula:  Distilled  water,  95  c.  c.;  nitric  acid  (strong), 
5  c.  c.;  picric  acid,  to  saturation.  Only  to  be  recommended  for  eggs  rich  in  yolk 
and  where  a  high  power  of  penetration  is  required. 

Fix  4  to  12  hours,  transfer  to  67%  alcohol,  1  day  82%  alcohol  several  days 
changed  frequently.  It  is  necessary  to  wash  out  thoroughly  which  is  accomplished 
however,  with  difficulty. 

§  22.  Perenyi's  fluid.  Formula:  10%  aq.  sol.  nitric  acid,  4  parts;  95% 
alcohol,  3  parts;  K%  aq.  sol.  chromic  acid,  3  parts.  An  embryological  fixer 
of  much  value.  It  is  also  serviceable  for  general  work. 

Fix  tissue  for  4  to  5  hours,  place  in  67%  alcohol  for  1  day,  82%  alcohol  several 
days. 

§  23.  Alcohol.  95%,  67  or  70%  alcohol.  The  employment 

of  most  of  the  fixers  so  far  mentioned  requires  the  expenditure  of  considerable 
time,  rendering  them  inapplicable  or  unsuitable  in  many  instances.  95%  alcohol 
itself  answers  admirably  for  most  histologic  objects,  fixing  well,  hardening  and 
likewise  dehydrating  (§  49)  preparatory  to  imbedding  in  paraffin  or  collodion, 
affording  thus  a  considerable  economy  of  time.  It  is  also  most  serviceable  in 
pathological  tissues  where  the  presence  of  bacteria  is  suspected.  In  some  instances 
67%  alcohol  answers  as  well  or  better,  while  in  other  cases  absolute  (99%)  alcohol 
should  be  employed. 

Fix  in  95%  alcohol  for  1  to  3  days,  changing  two  or  three  times,  after  3  or  4 
hours  and  after  24  hours.  The  tissue  will  probably  be  found  to  be  dehydrated 
and  ready  for  the  next  step  of  the  imbedding  process  (§  51  or  62).  Stain  as  you 
wish. 

§  24.  Alcohol-acetic.  The  addition  of  5%  glacial  acetic  acid  to  95% 
alcohol  or  absolute  alcohol  increases  the  penetrating  power  and  improves  the 
cutting  quality  of  objects  containing  much  connective  tissue.  The  following 
formula  possesses  high  penetration  and  is  sometimes  useful: 

§  25.     Alcohol-acetic  (Carnoy's  fluid,  3-1).     Formula:     Glacial  acetic  acid 

1  part,  95%  or  absolute  alcohol,  3  parts.     Transfer  after  fixing  to  95%  or  absolute 
alcohol,  changing  each  day  until  the  acetic  acid  is  well  washed  out. 

§  26.  Chloroform-alcohol-acetic.  (Carnoy's  6-3-1  ).  Formula:  95%  or 
absolute  alcohol,  6  parts;  chloroform,  3  parts;  glacial  acetic  acid,  1  part.  Fix 

2  to  24  hours  or  longer,  transfer  to  95%  alcohol,  changed  two  or  three  times. 

§  27.  Carnoy's  Sublimate  mixture.  The  above  (§  26)  with  mercuric  chlorid 
added  to  saturation.  A  very  penetrating  and  excellent  fixer,  particularly  useful 
for  nuclear  preservation  and  for  embryos. 

Fix  1  to  24  hours,  transfer  to  95%  alcohol  for  1  day,  then  to  82%  alcohol  for 
several  days,  changing  daily  and  adding  iodine  solution  (§11)  until  the  excess 
mercuric  chlorid  is  removed. 

§  28.  Alcohol-acetic-formol.  Combinations  such  as  the  following  are 
useful  for  general  purposes.  V.  Luko's  formula:  Alcohol,  67%,  or  82%,  100  c.  c. 
glacial  acetic  acid,  5  c.  c.;  formalin,  5-10  c.  c. 

Fix  for  12  to  24  hours  or  longer;  remove  to  67%  or  82%  alcohol  for  1  day 
or  more. 

§  29.  Formaldehyde.  Solutions  of  this  chemical  have  been  found  to  be 
good  preservatives  and  hardeners  and  fairly  good  fixers.  It  penetrates  rapidly, 


15 

and  preserves  the  natural  transparency  and  pigmentation  of  the  tissue,  making  it 
valuable  for  gross  anatomy  and  museum  purposes.  As  a  fixer,  an  aqueous  solu- 
tion of  2  to  4%  strength  may  be  employed,  or  it  may  be  used,  which  is  preferable, 
in  conjunction  with  other  chemicals,  as  picric  acid,  in  picro-formalin,  or  chromic 
acid  and  acetic  acid. 

Formalin  is  a  36  to  40%  solution  of  formaldehyde  (gas)  in  water.  A  small 
amount  of  formic  acid  is  also  present.  A  10%  solution  of  formalin,  that  is  a  4% 
solution  of  the  formaldehyde  is  a  satisfactory  strength  for  most  histological 
purposes. 

Fix  12  to  24  hours,  remove  to  67%  alcohol  for  a  day,  82%  alcohol  one  to 
several  days.  Stain  as  you  wish. 

§  30.  Osmic  acid.  A  very  useful  as  well  as  expensive  reagent  and  somewhat 
difficult  to  use.  It  is  generally  employed  as  a  fixer  in  conjunction  with  other 
reagents,  as  in  the  mixtures  (§  §  18  and  19).  When  used  alone  as  a  fixer  weak 
solutions  are  generally  best — A  to  1%.  It  penetrates  slowly  and  it  "over-fixes" 
cells  very  easily,  obscuring  detail  and  giving  the  parts  a  homogeneous,  glassy 
appearance.  Over-fixed  cells  cannot  be  stained,  or  with  great  difficulty.  More 
or  less  blackening  of  the  protoplasm  also  occurs.  It  may  be  used  chiefly  to  demon- 
strate fat,  which  is  blackened  by  it,  and  the  zymogen  of  pepsin  and  trypsin,  which 
it  preserves  and  browns  slightly. 

Fix  small  (about  2  mm.  thick  or  less)  pieces  of  tissue  in  1%  osmic  acid  for  6  to 
12  hours,  wash  well  in  water  (running  or  changed  frequently)  for  12  to  24  hours, 
and  place  in  67%  and  82%  alcohols.  It  is  somewhat  difficult  to  prevent  pure 
osmic  acid  of  this  strength  from  over-fixing  the  tissue,  and  cell  detail  is  generally 
lost,  though  the  form  of  cells  is  well  preserved. 

§  31.  Nitric  acid.  A  10%  solution  of  nitric  acid  is  serviceable  in  fixing  the 
blastoderm  of  the  chick. 

§  32.  Muller's  fluid.  Formula:  Potassium  dichromate,  2.5  grams;  sodium 
sulphate,  1  gram;  water,  100  c.  c.  Make  up  from  stock  solutions.  This  fluid  is 
more  of  a  hardener  than  a  fixer;  it  should  be  avoided  (as  likewise  Erlicki's  fluid 
and  potassium  dichromate)  when  the  preservation  of  nuclear  structure  is  desired. 
Staining  after  its  use  is  sometimes  difficult.  It  is,  however,  occasionally  useful 
for  general  work,  although  such  formulas  as  Zenker's  fluid  or  Kelly's  fluid  are 
generally  to  be  preferred. 

Place  the  object  in  an  abundance  of  the  fluid  and  harden  for  from  1  to  8 
weeks,  changing  the  fluid  at  first  each  day.  In  general,  10  to  14  days  will  be 
sufficient.  Wash  in  running  water  for  24  to  48  hours  or  longer,  remove  to  67% 
alcohol  for  1  to  2  days,  82%  alcohol  several  days.  Keep  in  the  dark  while  in  the 
alcohols,  and  change  to  fresh  when  the  fluid  is  colored  yellow.  Tissue  hardened 
in  Muller's  fluid  cuts  well,  and  it  is  useful  in  preparing  sections  of  large  organs,  or 
organs  with  much  connective  tissue.  Its  chief  usefulness  is,  however,  in  the  study 
of  the  nervous  system  (§  199-). 

§  33.  Erlicki's  fluid.  Formula:  Potassium  dichromate,  2.5  grams;  copper 
sulphate,  1  gram;  water,  100  c.  c.  Make  up  from  stock  solutions.  This  is  quite 
similar  to  Muller's  fluid  in  its  action  and  results,  save  that  its  action  is  more  rapid 
and  stronger.  Therefore,  it  had  better  be  employed  with  smaller  objects,  and 
allowed  to  act  only  2  to  14  days.  Otherwise,  employ  like  Muller's  fluid. 


16 

§  34.  Potassium  dichromate.  2%,  3%,  and  5%  aqueous  solutions.  This 
is  quite  similar  to  Miiller's  fluid  in  its  action,  and  may  be  employed  in  the  same 
cases.  It  is  generally  used  for  the  central  nervous  system. 

Harden  in  an  abundance  of  the  solution  for  2  to  8  weeks,  beginning  with 
the  2%  solution  for  2  to  6  days,  3%  solution  1  to  3  weeks,  5%  solution  1  to  3 
weeks.  Wash  out  in  running  water  24  to  48  hours.  Place  in  67%  and  82% 
alcohols  several  days,  keeping  in  the  dark  meanwhile,  changing  when  the  alcohol 
is  colored. 


ISOLATION. 

§  35.  One  of  the  simplest  ways  of  examining  the  structure  of 
a  tissue  is  the  separation  from  one  another  of  the  structural  elements 
composing  it,  thus  permitting  its  analysis.  Likewise,  for  a  correct 
conception  of  the  forms  of  the  cells  and  fibers  of  the  various  tissues 
of  the  body,  one  must  see  these  elements  isolated  and  thus  be  able 
to  inspect  them  from  all  sides.  It  frequently  occurs  also  that  isola- 
tion is  not  quite  complete  and  one  can  see  in  the  clearest  manner 
the  relations  of  the  cells  or  fibers  to  one  another. 

In  the  employment  of  this  method  the  tissue  may  be  taken 
fresh  and  isolation  accomplished  by  teasing  with  needles  or  similar 
instruments;  or  it  may  be  treated  with  media  which  will  serve  to 
render  teasing  partially  or  entirely  unnecessary.  In  such  cases 
simply  shaking  or  gently  tapping  the  preparation  will  often  suffice. 
In  many  instances  it  is  desired  to  examine  the  tissue  while  the 
elements  are  still  alive,  as,  for  example,  in  the  study  of  ciliated 
cells,  and  recourse  must  be  had  to  some  "normal,"  "indifferent" 
medium.  Best  of  all  is  the  medium  with  which  they  are  bathed  dur- 
ing life — in  the  case  of  tissue  from  the  animal  body,  blood  serum, 
the  aqueous  humor  of  the  eye,  liquor  amnioticus,  or,  as  an  artificial 
substitute  often  more  convenient  if  reagents  are  to  be  used  subse- 
quently, physiological  or  normal  salt  solution,  being  a  6/10  to  9/10% 
solution  of  common  salt  (sodium  chlorid)  in  distilled  water.  6  /10% 
is  suitable  for  use  with  Amphibian  tissue;  75/100%  is  normal  for 
reptiles  and  9/10%  best  for  mammals.  Other  normal  physiological 
solutions  may  in  some  cases  be  preferable,  such  as  Ringer's  solution, 
Locke's  solution,  etc. 

§  36.  The  use  of  chemical  solutions  to  facilitate  isolation  of  the 
elements  is  called  Maceration.  The  chemical  agents  or  solutions  for 
isolating  are,  in  general,  the  same  as  those  used  for  fixing  and  hard- 
ening. But  the  solutions  are  only  about  one-tenth  as  strong  as  for 


17 

fixing  and  the  action  is  very  much  weaker  and  requires  from  one  or 
two  hours  to  as  many  days.  In  the  weak  solution  the  cell  cement  or 
connective  tissue  is  softened  so  that  cells  and  fibers  may  be  separated 
from  one  another,  and  at  the  same  time  the  cells  are  preserved.  In 
other  words,  a  weak  fixing  action  is  retained  while  the  hardening 
action  is  reduced  on  dilution.  The  time  required  for  the  action  of 
the  dissociator  varies  inversely  as  the  vehemence  of  the  fixer  and 
the  density  of  the  tissue,  2  to  3  hours  to  several  days.  In  fixing  and 
hardening,  on  the  other  hand,  the  cell  cement,  like  the  other  parts  of 
the  tissue,  is  made  firmer.  It  is  better  also  to  dilute  the  fixing  agents 
with  normal  salt  solution  than  merely  with  water  [15].  Those 
chemicals  that  are  "cytoplasmic  fixers"  such  as  potassium  dichro- 
mate,  Muller's  fluid,  formaldehyde,  osmic  acid,  appear  to  be  especi- 
ally useful  in  the  dissociation  of  epithelia. 

For  the  isolation  of  muscle,  alkalis  or  mineral  acids,  which  soften 
or  dissolve  the  connective  tissue  are  to  be  employed.  Horn,  nail  and 
hair  require  strong  mineral  acid  or  (usually)  weak  alkali. 

§  37.  Of  the  many  maceration  fluids  or  dissociators  the  follow- 
ing may  serve  most  of  the  needs  of  histology:  (1)  Muller's  fluid  dis- 
sociator; (2)  formaldehyde  dissociator;  (3)  osmic  acid  (1/10%); 
(4)  sulphuric  acid  (strong);  (5)  nitric  acid  dissociator;  (6)  caustic 
potash  dissociator. 

§  38.  Muller's  fluid  dissociator.  Formula:  Muller's  fluid,  1 
part;  physiological  salt  solution,  9  parts  (i.  e.,  potassium  dichromate, 
2.5  grams;  sodium  sulphate,  1  gram;  sodium  chlorid,  9  grams; 
water,  1,000  c.  c.)%  [15]. 

This  is  a  good  dissociator  for  epithelia,  including  glands.  Dilution 
decreases  the  hardening  action  of  Muller's  fluid  as  is  shown  especially 
on  the  cell-cement, — hence  its  dissociating  action.  Considerable 
latitude  in  time  is  allowed  in  the  use  of  this  dissociator;  12  hours 
being  often  sufficient,  although  a  stay  of  several  days  in  the  dis- 
sociator usually  does  no  harm. 

§  39.  Directions  for  use.  In  the  employment  of  this  fluid  for 
the  isolation  of  epithelial  cells,  proceed  as  follows: 

Place  the  tissue  covered  with  the  epithelium  which  it  is  desired 
to  isolate  in  the  dissociator  in  a  shell  vial  or  dish,  where  it  may  re- 
main from  2  to  3  hours  to  2  to  3  days;  for  the  epithelium  of  the  tra- 
chea, intestines,  etc.,  the  action  is  sufficient  in  2  to  3  hours,  although 
good  preparations  may  be  obtained  after  two  days  or  more.  For  the 


18 

stratified  epithelia,  like  those  of  the  skin,  mouth,  etc.,  it  may  require 
1  to  3  days  for  the  most  satisfactory  preparations.  After  the  tissue 
has  remained  in  the  dissociator  a  sufficient  time,  scrape  the  epithelial 
surface  gently  with  a  scalpel  and  place  the  scrapings  on  a  slide  in  a 
drop  of  dissociator;  cover  and  examine.  If  one  proceeds  after  two 
hours  or  so,  probably  most  of  the  cells  will  cling  together,  and  in 
the  various  clumps  will  appear  cells  on  end  showing  the  tops  or 
bases,  and  other  clumps  will  show  the  cells  in  profile. 

Tap  the  cover  gently  with  a  needle-holder  or  other  light  object 
in  order  to  separate  the  cells  from  each  other  more  completely. 
Many  fully  isolated  cells  as  well  as  cells  in  groups  will  be  seen.  Ex- 
amine carefully. 

§  40.  Staining.  Scrape  gently  the  epithelial  surface  in  a  fresh 
spot  and  place  the  scrapings  on  the  slide  in  a  drop  of  eosin  (§  114) 
or  congo  red  (§  118).  Mix  well  so  that  the  stain  can  penetrate.  If 
for  temporary  examination,  cover  immediately  and  examine  as  be- 
fore. For  methods  of  making  permanent  preparations  of  dissociated 
cells  and  mounting  in  glycerin,  see  §  §  151—. 

§  41.  Formaldehyde  Dissociator.  Formula:  40%  formaldehyde 
(formalin),  2  c.  c.;  physiological  salt  solution,  1,000  c.  c.  (i.  e., 
.08%  sol.  of  formaldehyde  in  normal  salt  solution).  This  is  a  good 
general  dissociator  and  as  such  may  be  employed  instead  of  Miiller's 
fluid  dissociator.  It  is  especially  serviceable  in  the  isolation  of  the 
nerve  cells  of  the  brain  and  spinal  cord  [13],  and  for  its  use  with 
that  material,  see  §  §  151—.  It  is  however  excellent  for  the  dissocia- 
tion of  epithelia. 

§  42.  Osmic  acid.  A  1-10%  solution  of  osmic  acid  is  a  valua- 
ble dissociator,  especially  serviceable  in  the  isolation  of  nerve-fibers, 
my  clinic  and  amyelinic,  and  when  fat  is  present,  since  fat  and  the 
myelin  of  myelinic  nerve-fibers  are  blackened  by  it.  Twelve  to  twen- 
ty-four hours  generally  affords  sufficient  time  for  it  to  act.  (§  193, 


§  43.  Sulphuric  Acid.  This  is  used  in  the  concentrated  form 
as  a  dissociator  of  the  epithelial  cells  of  hair,  horn,  and  nail.  If 
heated,  a  few  minutes  suffice;  employed  cold,  a  day  or  two  may  be 
required. 

§  44.  Nitric  acid  dissociator.  [8]  Formula:  Strong  nitric 
acid,  20  c.  c.;  water,  80  c.  c.  See  §  189a.  This  fluid  is  employed  in 
the  isolation  of  muscle  fibers,  both  striated  and  plain. 


19 

§  45.  Caustic  potash  dissociator.  [8]  Formula:  Caustic 
potash,  potassium,  hydroxid  (in  sticks),  35  to  40  grains;  distilled 
water,  65  or  60  c.  c.  This  solution  will  be  used  for  the  isolation  of 
cardiac  muscle  'cells',  although  it  may  be  used  for  striated  or  plain 
muscular  tissue,  or  as  a  general  dissociator.  It  may  also  be  employed 
for  isolating  the  cells  of  hair,  horn  or  nail,  either  full  strength  or 
diluted. 

Ten  to  fifteen  minutes  or  longer  will  be  enough  for  the  isolation 
of  heart  muscle  (§  189  6) ;  2  to  3  days  may  be  required  for  the  ade- 
quate maceration  of  cornified  epithelial  cells. 


SECTIONING. 

§  46.  In  addition  to  the  examination  of  tissue  by  the  separa- 
tion of  the  component  elements — isolation — it  may  be  examined 
microscopically  after  cutting  very  thin  slices  or  sections  of  it.  This 
may  be  done  free-hand  or  by  means  of  a  special  machine,  a  micro- 
tome, and  with  or  without  an  imbedding  and  supporting  mass. 

For  the  finer  work  an  imbedding  mass  and  a  microtome  must 
be  used.  Free-hand  sectioning  without  an  imbedding  mass,  and 
even  without  previous  fixing  is,  however,  necessary  or  advisable 
when  economy  of  time  is  a  desideratum,  as  in  clinical  examinations 
of  tissue,  when  one  wishes  to  study  the  part  alive  or  fresh  (i.  e., 
not  treated  with  reagents),  or  if  the  reagents  necessary  for  fixing 
and  imbedding  destroy  or  alter  the  structural  features  to  be  investi- 
gated. 

The  ability  to  recognize  tissues  and  organs  unaffected  by  re- 
agents and  without  the  employment  of  methods  involving  the  ex- 
penditure of  time  and  effort  is  very  desirable,  especially  in  patho- 
logical work,  when  haste  often  forbids  the  employment  of  the  finer 
methods,  were  facilities  for  their  use  available,  as  in  some  cases 
they  are  not.  Great  skill  in  the  use  of  simple  tools  may  be  gained 
and  counts  for  much.  It  should  be  remembered  also  that  the  greater 
one's  knowledge  of  a  structure  the  less  the  need  to  resort  to  special 
methods  of  preparation  for  its  recognition. 


IMBEDDING   METHODS. 

§  47.     When  the  consideration  of  time  is  not  so  important  and 
finer  results  are  more  to  be  desired,  the  sections  should  be  prepared 


20 

according  to  some  method  in  which  an  imbedding  mass  is  used.  The 
interstices  of  the  tissue  are  completely  filled  with  some  substance 
that  wrill  give  support  and  greater  consistency  and  homogeneity  to 
the  tissue,  and  thereby  enable  the  cutting  of  much  thinner  and  more 
perfect  sections. 

There  are  three  methods  that  are  generally  employed,  (a)  the 
Paraffin  method,  (b)  the  Celloidin  (Collodion)  method,  and  (c)  the 
Freezing  method;  the  imbedding  masses  to  fill  the  spaces  being  re- 
spectively paraffin,  collodion  and  a  congelation  mass,— ice.  The  last 
is  the  simplest;  it  requires  less  expenditure  of  time,  fewer  reagents, 
and  its  results  are  in  some  ways  the  crudest.  As  in  cutting  free-hand 
sections  without  imbedding,  the  freezing  microtome  should  be  em- 
ployed when  haste  is  necessary  and  finer  detail  unimportant,  as  in 
clinical  work.  The  two  remaining  methods  may  be  employed  in  most 
cases  and  give  good  results.  A  choice  between  them  mUst  be  deter- 
mined by  the  special  requirements  of  the  case  and  a  consideration  of 
the  differences  of  the  two  methods  as  set  forth  in  tabular  form  below : 

CELLOIDIN.  PARAFFIN. 

No  heat  required.  Heat  required. 

Sections  relatively  thick ;    10  to  25  mi-  Sections  relatively  thin ;   1  to  10  microns 

crons  or  more.  or  more. 

Imbedding  mass  usually  not  removed.  Imbedding  mass  removed. 

Sections  usually  cut  wet  (with  alcohol  or  Sections  cut  dry. 

oil). 

Knife  oblique.  Knife  usually  set  at  right  angles. 

Cutting  stroke  slow.  Cutting  stroke  usually  rapid. 

Form  of  the  organ  better  preserved.  Usually  more  or  less  distortion. 

Imbedding  requires  more  time.  Imbedding  requires  less  time. 

Celloidin  stains  with  basic  dyes.  

In  general,  better  for  larger  specimens.  Better  for  smaller  objects. 

§  48.  Despite  the  differences,  the  two  methods  may  in  most 
cases  be  used  interchangeably.  The  advantages  of  the  paraffin 
method  are  (1)  the  facility  of  its  use  and  the  ease  with  which  thin 
sections  may  be  obtained  and  its  adaptation  to  serial  sectioning. 
Celloidin  is  particularly  useful  when  heal  is  injurious  or  the  paraffin 
solvents  (clearers)  dissolve  out  substances  which  it  is  desired  to 
preserve.  Its  main  defect  is  the  readiness  with  which  the  celloidin 
stains  with  basic  stains,  particularly  such  as  Iron  Hematoxy lin  and 
the  coal-tar  dyes  (safranin,  gentian  violet,  methylene  blue,  etc.) 
The  celloidin  may,  however,  be  dissolved  out.  See  §  136. 

§  49.  The  following  table  will  indicate  the  steps  in  the  employ- 
ment of  the  two  methods: 


Paraffin  Method 


21 
Living  tissue 

( 

Fixing 

(§  §  i-) 

Washing 

i 
Alcohols  (§  9) 

(50%,  67%,  82%)<- 

Dehydration 
(95%-99%  alcohol) 


-Staining  in  toto 
(§79) 


Celloidin  Method 


Toluene,  1   part 
Abs.  Alcohol,  1  part. 
(Ihr.  to  1  day) 

1 
Pure  toluene, 

(1  hr.  to  1  day) 

i 

Toluene,  1  part, 
Paraffin,  1  part. 
(2  hrs.  to  2  days) 

i 

Pure  Paraffin 

(2 -to  24  hrs.) 


Clearing 


Ether-alcohol 
days) 


.  .  Infiltration 


Imbedding  paraffin, 

mass  cooled.         Imbedding. 


Paraffin  sections 
cut. 


Sectioning 


Thin  celloidin 

(2%  solution) 

(1-14  days) 

f: 

Thick  celloidin 
(6%  solution) 
(1-14  days) 

1 
Thick  celloidin 

(12%  solution) 
(1-14  days) 

!  - 

Celloidin  mass 
hardened  in 
chloroform 

! 

Celloidin  block 
clarified  or 
placed  in  82% 
alcohol 


Celloidin  sections 
.  .cut 


22 

THE    PARAFFIN    METHOD. 

§  50.  As  seen  by  the  above  scheme,  the  aim  is  to  fill  all  the 
interstices  of  the  tissue  with  paraffin  of  the  right  degree  of  hardness 
to  have  it  cut  well.  Paraffin  is  not  soluble  in  water  or  alcohol,  but 
is  soluble  in  a  number  of  fluids  which  in  turn  are  miscible  with  alco- 
hol. Hence  the  following  steps  are  necessary:  (1)  the  tissue  must 
be  first  water-rid,  thoroughly  dehydrated  with  strong  alcohol;  (2) 
freed  from  the  alcohol,  cleared  by  a  fluid  that  mixes  with  melted 
paraffin  which  (3)  takes  the  place  of  the  clearer  in  the  tissue,  infil- 
trates it,  filling  the  spaces;  (4)  finally,  the  tissue  is  imbedded  in 
paraffin  of  the  right  degree  of  hardness,  the  mass  cooled,  and  it  is 
ready  (5)  to  cut,  or  section. 

§  51.  Dehydration.  After  the  various  steps  pertaining  to  the 
fixing  and  hardening  (§  §  7,  9)  of  the  tissue  have  been  properly 
pursued  it  may  be  stored  in  alcohol  of  82%  to  95%  strength  depend- 
ing on  the  tissue  and  its  purpose.  The  dehydration  necessary 
in  imbedding  may  be  accomplished  by  immersion  in  alcohol  of  95% 
strength.  For  most  work  it  is  perhaps  better  to  employ  stronger 
(absolute)  alcohol.  If  xylene  or  toluene  are  used  for  clearing  absolute 
alcohol  must  be  used. 

Immerse  small  pieces  2  to  3  mm.  in  diameter  for  at  least  6  to  8 
hours  in  95%  alcohol  changed  once  or  twice.  A  longer  time,  even 
days,  usually  does  no  harm  and  is  preferable  to  ensure  complete 
dehydration.  For  larger  pieces  of  tissue  or  entire  organs  a  corres- 
pondingly longer  period  of  dehydration  should  be  employed,  a  several 
days'  stay,  with  the  alcohol  changed  daily,  being  often  advisable.  In 
any  case,  dehydrate  thoroughly,  changing  the  alcohol  1  to  3  times,  the 
last  change  usually  being  to  absolute  alcohol.  Let  the  tissue  dehy- 
drate for  a  longer  rather  than  a  shorter  period  of  time. 

§  52.  Clearing.  The  alcohol  must  next  be  replaced  by  some 
solvent  of  paraffin  which  is  miscible  with  alcohol, — a  step  spoken  of 
as  clearing.  Toluene*  is  one  of  the  most  serviceable  clearers,  although 
for  special  purposes  other  media  such  as  xylene,  cedarwood  oil, 
bergamot  oil  or  chloroform  may  be  preferred.  Toluene  (and  xylene) 
mix  well  only  with  absolute  alcohol,  hence  the  dehydration  needs  to 
be  thorough,  and  the  clearing  and  infiltrating  is  best  accomplished  by 
a  number  of  steps.  After  the  absolute  alcohol,  the  tissue  is  placed  (1) 


*Benzene,  toluene,  xylene,  etc.;    the  terms  adopted  by  American  chemists. 
In  Europe  the  same  substances  are  designated  xylol,  benzol,  etc. 


23 

in  equal  parts  of  toluene  and  absolute  alcohol  for  1  hour  to  1  day;  (2) 
this  is  replaced  by  pure  toluene  for  an  equal  period  when  the  tissue 
will  be  clear  and  translucent, — except,  of  course,  such  as  is  dark  in 
color. 

Xylene  may  be  used  in  place  of  toluene  in  nearly  all  cases.  Steps  1  and  3  may 
often  be  omitted  with  very  small  objects.  In  some  cases  it  is  well  to  clear  with 
cedarwood  oil  first  (§  54)  and  then  transfer  to  toluene  (or  xylene),  toluene  paraffin, 
etc.  Familiarity  with  the  tissue  and  the  reagents  will  permit  considerable  depart- 
ture  from  a  fixed  line  of  procedure. 

§  53.  Infiltration.  After  the  tissue  is  completely  cleared  in 
the  toluene,  remove  it  to  a  dish  of  (3)  melted  infiltration  paraffin 
1  part,  toluene  1  part,  and  set  it  in  a  warm  place  (about  38°  C.),  so 
that  the  paraffin  may  remain  melted  and  the  toluene  slowly  evaporate. 
After  several  hours  or  even  days  with  very  large  pieces  place  the  tissue 
in  (4)  pure  paraffin  in  the  paraffin  oven  for  2  to  24  hours,  depending  on 
the  size  of  the  piece.  Quite  large  pieces  may  be  left  longer ;  with  them 
one  or  two  changes  to  fresh  infiltration  paraffin  may  be  necessary. 
The  melted  paraffin  replaces  the  toluene,  filling  in  the  interstices  of  the 
tissue.  Paraffin  melting  at  about  52-54°  C.  is  used.  It  is  best  not  to 
expose  to  a  higher  temperature  than  is  necessary,  or  for  a  long  period 
of  time  as  the  heat  tends  to  shrink  and  toughen,  especially  if  the  dehy- 
dration (and  consequently  the  clearing)  have  been  incomplete;  this 
is  particularly  true  of  organs  rich  in  connective  tissue.  The  paraffin 
oven  wTill  be  maintained  at  a  temperature  of  54-5°  C. 

§  54.  Other  Clearers.  Cedarwood  oil  is  a  good  clearer.  It  will  clear  from 
95%  alcohol  if  the  dehydration  has  been  carefully  done.  The  tissue  should  be  left 
in  the  cedarwood  oil  until  it  sinks  and  the  alcohol  currents  have  ceased  to  rise  from 
it.  The  steps  are  given  below.  Thickened  cedarwood  oil  such  as  is  used  for 
immersion  objectives  has  been  found  to  give  excellent  results  with  small  objects 
and  is  to  be  recommended  for  some  cell  work.  Chloroform  gives  excellent  results 
but  it  penetrates  (clears)  slowly,  so  that  it  should  be  used  only  with  small  pieces 
of  tissue.  Chloroform  or  thickened  cedarwood  oil  is  to  be  employed  with  objects 
in  which  it  is  desired  to  preserve  fat  which  has  been  blackened  by  osmic  acid 
(§  227). 

CHLOROFORM.  CEDARWOOD    OIL. 

Alcohol  (97-99%) .  95%  or  absolute  alcohol. 

Chloroform,  until  it  sinks  below  the  sur- 
face. 

Chloroform  and  paraffin,  equal  parts, 
for  4-48  hours,  at  incubator  tempera- 
ture (38  C.) .  cedarwood  oil. 

Pure  paraffin,  in  the  paraffin  oven.  Pure  paraffin,  in  paraffin  oven. 


24 

§  55.  Imbedding.  It  is  best  to  use  fresh  paraffin  for  imbed- 
ding and  sometimes  with  a  melting  point  higher  than  that  of  the  in- 
filtration paraffin, — 52  to  54°  C.  paraffin,  answers  well  in  a  room  of  19° 
to  20°  C.,  and  will  be  generally  used.  If  the  cutting  is  to  be  done  in 
a  room  of  lower  temperature,  a  softer  grade  of  paraffin  may  be  used 
for  imbedding;  if  at  a  higher  temperature,  a  harder  paraffin  should  be 
chosen,  as  when  summer  work  is  necessary. 

As  a  general  rule,  hard  tissues  require  a  harder  imbedding  par- 
affin which  is  also  better  when  very  thin  sections  are  desired.  Large 
sections  which  usually  must  also  be  relatively  thicker  need  a  softer 
paraffin.  It  is  better  to  work  with  a  paraffin  harder  than  the  room 
temperature  itself  would  call  for  and  then  regulate  the  cutting  tem- 
perature by  placing  a  source  of  heat  such  as  an  electric  light  nearer 
or  farther  away  from  the  microtome  knife. 

Make  a  small  paper  box,  fill  it  with  the  melted  imbedding  paraffin; 
float  the  box  on  a  dish  of  cold  water;  transfer  to  it  the  tissue  from  the 
paraffin  oven,  arrange  it  carefully  in  the  box  in  the  way  you  wish  it 
for  cutting,  and  let  the  mass  cool. 

§  56.  In  imbedding  in  paraffin  observe  the  following  rules: 
(1)  Take  no  more  paraffin  (no  larger  box)  than  is  needed  to  form  a 
mass  of  convenient  size  around  the  specimen.  The  aim  is  to  have 
as  homogeneous  a  mass  as  possible;  paraffin  tends  to  crystallize  if 
it  cools  slowly,  hence  the  smaller  the  mass  the  more  rapidly  may  it 
be  cooled.  (2)  Let  the  imbedding  paraffin  when  poured  into  the 
box  be  several  degrees  above  its  melting  point,  and  the  tissue  like- 
wise should  have  an  equal  temperature.  Should  the  imbedding 
paraffin  (or  the  tissue)  be  too  cool  it  will  not  set  well  around  the 
specimen,  and  a  film  of  air  may  be  enclosed.  On  the  other  hand, 
take  care  that  the  paraffin  is  not  hot  enough  to  "cook"  the  tissue, 
thereby  shrinking  it  and  rendering  it  hard  and  tough  or  ruining  it 
altogether.  (3)  Cool  the  paraffin  by  floating  the  box  on  cold  water. 
A  homogeneous,  translucent,  paraffin  mass  can  only  be  secured  if  it 
is  quickly  cooled.  When  a  film  has  formed  on  the  surface  strong 
enough  to  resist  rupture,  immerse  the  block,  or  drop  95%  alcohol 
upon  the  surface.  Ice  is  an  advantage  in  summer  imbedding  if  cold 
water  is  not  available.  When  ice  or  cold  water  are  not  available, 
good  results  have  been  secured  by  floating  the  box  on  a  shallow  dish 
of  (used)  ether-alcohol  (§  63).  A  homogeneous  paraffin  is  only 
secured  if  the  paraffin  is  allowed  to  shrink  in  cooling;  it  is  therefore 
well  to  make  the  boxes  as  shallow  as  possible, — that  is,  much  broader 


25 

and  longer  than  high.     Watch  glasses,  watch  crystals,  small  tin  pans, 
etc.,  may  be  used  as  imbedding  receptacles. 

§  57.  Crystallization  of  the  imbedding  mass.  Paraffin  that  has  crystallized 
is  crumbly  and  will  not  give  good  sections.  When  crystallization  has  occurred, 
it  is  best  to  reimbed.  Its  occurrence  is  usually  due  to  too  slow  a  cooling  of  the 
imbedding  mass,  or  cooling  under  conditions  that  prevent  the  paraffin  from  shrink- 
ing. It  is  sometimes  due  to  the  presence  of  impurities  in  the  paraffin,  such  as 
water  (?),  excess  of  clearer,  etc. 

§  58.  Cutting  the  sections.  The  essentials  for  good  paraffin 
sectioning  are  (1)  well-imbedded  tissue,  (2)  a  sharp  microtome  knife 
(or  section  razor),  (§  173),  (3)  a  room  of  the  proper  temperature,  and 
(4)  the  paraffin  block  properly  trimmed  and  arranged  in  the  micro- 
tome. Furthermore,  tissues  fixed  and  hardened  in  different  ways  cut 
very  differently.  Tissue  fixed  in  Hermann's,  Flemming's,  Mailer's, 
Zenker's  fluid  or  Carnoy's  fluid,  etc.,  cuts  well;  alcohol  and  mercuric 
chlorid  tissue  is  more  apt  to  be  tough  or  hard,  etc.  The  different 
organs  and  tissues  have  of  course  very  different  adaptabilities  to  the 
method. 

After  the  imbedding  mass  is  well  cooled,  remove  the  paper  box 
and  trim  the  part  containing  the  tissue  in  a  pyramidal  form,  two  of 
the  sides  at  least  being  as  nearly  parallel  as  possible.  Clamp  the 
block  of  paraffin  in  the  holder  of  the  microtome  so  that  the  tissue 
will  be  at  the  proper  level  for  cutting,  being  careful  to  have  the  par- 
allel sides  also  parallel  to  the  edge  of  the  knife.  If  a  ribbon  micro- 
tome is  used,  heat  the  holder  and  melt  the  end  of  the  block  upon  it. 
Cool  and  place  the  holder  in  its  place  in  the  microtome,  again  hav- 
ing the  parallel  sides  and  the  edge  of  the  knife  parallel.  Use  a  very 
sharp,  dry  section  knife  for  cutting  the  sections.  Clamp  it  in  the 
microtome  slightly  inclined  to  the  cutting  surface  of  the  tissue.  If 
the  temperature  of  the  room  is  right  for  the  paraffin  used,  the  sec- 
tions will  remain  flat,  and  if  the  directions  given  above  for  trimming 
and  arranging  the  block  be  observed,  they  will  adhere  and  thus  form 
a  ribbon.  If  the  room  is  too  cold  or  the  paraffin  too  hard,  the  sec- 
tions will  roll;  if  it  is  too  warm,  the  sections  will  crush  or  be  imper- 
fect. If  a  microtome  in  which  the  knife  is  not  fixed,  is  used,  make 
the  sections  with  a  rapid  straight  cut  as  in  planing.  Do  not  try  to 
section  with  a  drawing  cut  as  used  in  celloidin  sectioning.  10^  will 
be  found  the  most  convenient  thickness  for  the  sections,  though  in 
special  cases  they  should  be  thinner  or  even  thicker.  Handle  the 
sections  by  means  of  a  camel's  hair  brush,  a  needle,  or  sometimes  on  a 
scalpel  handle,  when  cutting  ribbons,  etc. 


§  59.  Difficulties  in  sectioning,  (a)  Rolling  of  the  sections  indicates  too 
hard  a  paraffin,  tissue  or  both,  or  too  low  a  cutting  temperature;  bring  the 
source  of  heat  nearer  or  await  better  conditions,  (b)  Crushing  together  of  the 
sections  or  wrinkling.  Too  soft  a  paraffin,  too  high  a  temperature,  imperfect 
infiltration:  alter  conditions  or  reimbed,  or  reinfiltrate,  etc.  (c)  The  sections  do 
not  ribbon.  Due  usually  either  to  (a)  or  to  a  failure  to  have  the  two  sides  of  the 
block  trimmed  parallel  and  set  parallel  to  knife  edge,  (d)  The  sections  crack 
parallel  to  knife  edge:  tissue  very  brittle,  (e)  The  sections  are  electric.  The 
electricity  is  mainly  due  to  friction  in  the  cutting.  The  hardness  of  the  tissue, — 
in  some  cases  due  to  presence  of  metallic  salts, — or  imperfection  in  the  knife  edge 
seem  in  many  cases  to  be  responsible;  although  in  many  instances  the  cause 
appears  obscure.  A  tube  of  radium  or  a  strong  induction  coil  operating  in  the 
immediate  neighborhood  is  said  to  obviate  the  difficulty. 

Remember  to  have  the  paraffin  block  trimmed  with  two  sides 
parallel  and  the  knife  edge  parallel  to  these.  Also,  do  not  attempt 
to  cut  if  the  temperature  of  the  room  is  too  high, — above  23°  C. 

§  60.  Resume  of  the  method.  To  obtain  as  good  results  as 
possible  with  a  certain  organ  fixed  and  hardened  in  a  certain  way, 
the  steps  must  be  carefully  and  exactly  followed.  Let  the  dehydra- 
tion be  complete,  clearing  thorough,  infiltration  sufficient;  imbed, 
carefully  observing  the  three  cautions  mentioned;  and  in  cutting, 
remember  to  have  a  sharp  knife,  a  cool  room,  and  the  imbedding 
block  properly  trimmed.  Success  also  depends  largely  on  the  pre- 
vious treatment  in  the  fixer  and  on  the  care  with  which  the  fixer  is 
washed  out. 

Properly  employed,  the  paraffin  method  is  widely  serviceable, 
being  only  useless  where  the  tissue  is  very  large,  very  hard,  hard- 
ened or  injured  by  heat,  or  w^here  the  exact  form  of  a  large  organ  is 
important. 


THE    CELLOIDIN   METHOD. 

§  61.  A  comparison  with  the  paraffin  method  has  already  been 
given  (§  §  47,  48);  there  may  be  emphasized  here  three  points:  (1) 
writh  paraffin  heat  is  required,  with  celloidin  no  heat;  (2)  paraffin 
must  be  removed  from  the  sections  subsequently,  celloidin  need  not  be 
and  usually  is  not  dissolved  out;  (3)  by  the  paraffin  method  may 
be  obtained  small  sections  (5  cm.  square  or  less),  and  thin,  by  the 
celloidin,  larger  sections,  but  thicker.  With  paraffin  heat  (melting 
and  cooling)  is  necessary,  and  the  mass  is  sometimes  spoken  of  as  a 
fusion  imbedding  mass;  celloidin  is  a  solution,  and  the  mass  is  left 
in  the  tissues  by  evaporation,  or  its  equivalent. 


27 

In  the  celloidin  method  the  imbedding  mass  with  which  the 
spaces  of  the  tissue  are  to  be  filled  is  collodion,  a  solution  of  celloidin 
or  proxylin*  (soluble  cotton)  in  ether  and  alcohol,  hence  the  steps, 
which  are  comparable  with  those  of  the  paraffin  method  (see  §  47), 
are  (1)  Dehydration,  removal  of  the  water;  (2)  Saturation  with 
either-alcohol,  the  solvent  of  the  celloidin;  (3)  Infiltration  with  cel- 
loidin solutions,  a  thin  and  a  thick;  (4)  Imbedding  in  a  thick  cel- 
loidin mass,  which  is  hardened  and  (5)  sections  cut. 

§  62.  Dehydration.  Let  it  be  complete,  as  in  the  prepara- 
tion for  paraffin  imbedding  (§  51).  Immerse  the  tissue  in  95% 
alcohol  for  12  to  24  hours  or  longer,  changing  1  to  3  times.  Consult 
also  §  51  upon  the  dehydration  of  tissue. 

§  63.  Saturation  with  ether-alcohol  (equal  parts  of  pure  ether 
and  absolute  alcohol).  Remove  the  tissue  from  the  strong  alcohol 
and  place  it  in  a  stoppered  vial  of  ether-alcohol  for  12  to  24  hours.  In 
addition  to  preparing  the  tissue  for  the  collodion  solutions,  it  com- 
pletes the  dehydration,  should  it  be  imperfect.  In  special  cases,  or 
if  the  dehydration  is  very  thorough  and  the  specimen  small,  this  step 
may  be  omitted.  A  satisfactory  infiltration  is,  however,  more  cer- 
tain if  ether-alcohol  be  used. 

§  64.  Infiltration,  (a)  with  thin  celloidin.  Pour  off  the 
ether-alcohol  and  add  the  thin  (2%)  solution  of  celloidin  in  ether- 
alcohol.  This,  being  a  solution  in  ether-alcohol  with  which  the  tissue 
is  saturated,  readily  permeates  it.  It  is  best  to  allow  at  least  a  day 
for  this  to  take  place,  although  if  there  is  time  a  stay  of  several  days 
is  better,  there  being  little  or  no  danger  of  deterioration  while  in  the 
solution.  With  large  (1  c.  c.  +)  objects  an  infiltration  of  a  week  or 
even  a  month  is  advisable. 

Infiltration,  (b)  with  thick  celloidin.  Pour  off  the  thin  col- 
lodion solution  and  add  thick  (5  or  6%)  solution  (in  ether-alcohol). 
In  this  there  is  gradual  concentration  of  the  solution  in  the  tissue. 
Allow  small  specimens  to  remain  a  day,  or,  better,  several  days; 


*Celloidin  is  a  specially  prepared  and  purified  form  of  pyroxylin.  It  is- 
about  twice  as  expensive  as  pyroxylin  or  soluble  cotton  but  with  it  better  solutions 
can  be  prepared.  The  pyroxylin  on  the  market  seldom  affords  stronger  solutions 
than  8%;  with  celloidin  a  12%  solution  is  easily  prepared.  The  trimmings  from 
the  celloidin  blocks  (after  the  alcohol  or  chloroform  hardening)  may  be  dried  out 
and  redissolved  and  thus  used  over  and  over  again.  Pyroxylin  may  be  used 
equally  well. 


28 

larger  objects  should  be  given  a  proportionately  longer  time,  a  week 
to  a  month,  or  even  longer. 

If  the  object  to  be  imbedded,  such  as  many  embryological  speci- 
mens, is  one  with  large  interior  cavities  with  thin  walls  the  transfer 
from  the  thin  solution  to  the  thick  solution  may  be  attended  by  a 
collapse  of  the  walls  and  a  consequent  shriveling  and  distortion  of 
the  specimen.  Avoid  this  by  allowing  the  thin  solution  to  thicken 
very  gradually  by  evaporation  in  a  dry  atmosphere,  as  under  a  bell- 
jar  with  calcium  chlorid  present  until  the  solution  has  attained  the 
right  consistency.  To  accomplish  this  it  is  only  necessary  to  have 
the  cork  of  the  vial  containing  the  specimen  perforated  by  a  small 
hole.  A  small  piece  of  paper  may  be  inserted  with  the  cork,  or  with 
porous  corks  no  special  effort  need  be  made.  Unless  the  thick  solu- 
tion has  itself  thickened  by  evaporation,  with  large  specimens  it  is 
advisable  to  follow  the  6%  bath  with  a  stay  in  a  thicker  solution,  as 
10  or  12%,  for  a  day  or  so. 

§  65.  Imbedding.  Pour  off  the  6%  or  12%  solution  and  add  for 
a  short  time  at  least  a  12%  solution  of  celloidin  (in  ether-alcohol). 
The  tissue  is  now  ready  for  imbedding  in  12%,  which  may  be  ac- 
complished in  either  of  two  ways :  (a)  on  a  holder  or  (b)  in  a  paper 
box.  Only  those  specimens  need  be  imbedded  in  a  box  that,  from 
their  shape,  or  for  purposes  of  careful  orientation  or  serial  sectioning, 
require  a  larger  imbedding  mass  around  them. 

(a)  On  a  holder  (wooden-block).  Choose  a  block  of  a  conveni- 
ent size;  put  a  drop  or  two  of  celloidin  upon  one  end  and  insert  a 
pin  vertically  to  the  surface  near  the  edge.  Transfer  the  tissue  from 
the  vial  of  thick  celloidin  to  the  block  and  lean  it  against  the  pin. 
The  shape  of  many  tissues  will  obviate  the  need  of  a  pin.  Pour  the 
thick  celloidin  onto  the  tissue,  drop  by  drop,  moving  the  block  in 
such  a  way  that  the  thick  viscid  mass  may  be  made  to  surround  and 
envelop  the  tissue.  Continue  to  add  drops  of  celloidin  at  intervals 
until  the  tissue  is  well  surrounded,  and  then  as  soon  as  a  slight  film 
hardens  on  the  surface  invert  the  holder  bearing  the  tissue  in  a 
shell-vial  of  large  diameter  or  glass  box  containing  enough  chloroform 
to  cover  the  specimen.  Cork  or  cover  so  that  the  chloroform  will 
not  evaporate.  If  the  piece  of  tissue  is  of  awkward  size  and  shape, 
oiled  paper  may  be  wound  around  the  end  of  the  wooden  holder  and 
tightly  tied,  the  projecting  hollow  cylinder  being  long  enough  to 
receive  the  object.  The  tissue  may  be  put  into  the  cylinder  as  before, 
the  celloidin  slowly  poured  in  drop  by  drop  until  the  specimen  is 


29 

completely  covered.     When  a  film  has  formed,  place  in  chloroform 
as  before. 

(b)  In  a  paper  box.  When  a  box  is  required  for  imbedding 
proceed  as  follows:  The  inside  of  the  paper  box  should  be  slightly 
oily  to  prevent  the  celloidin  from  sticking  to  it.  Rub  upon  the 
paper  that  is  to  be  folded  to  form  the  box  a  little  vaseline,  and  then 
with  a  cloth  or  lens  paper  remove  as  much  as  possible.  Fold  the 
paper  into  a  box  of  convenient  size  and  shape.  Remove  the  object 
from  the  thick  celloidin  and  place  it  in  the  box,  arranging  it  in  the 
manner  wished  with  a  view  to  sectioning  it  later.  Pour  over  it 
slowly,  drop  by  drop  or  a  little  at  a  time,  a  12%  solution  of  celloidin 
until  the  specimen  is  well  covered  and  the  box  sufficiently  filled. 
It  is  better  to  have  a  deep  layer  over  the  specimen.  The  12%  solu- 
tion does  not  afford  the  best  mass  for  cutting,  so  that,  with  large 
objects,  it  is  better  to  allow  the  mass  in  the  box  to  thicken  by  evap- 
orating it  slowly  under  a  bell-jar  (aquarium  jar)  until  it  has  attained 
such  a  consistency  that  it  is  no  longer  fluid. 

§  66.  Hardening.  When  the  celloidin  mass  is  thick  enough 
so  that  it  only  dents  when  touched  with  the  finger  nail  it  is  ready  for 
hardening.  This  may  be  done  by  pouring  chloroform  into  the  jar 
in  which  the  imbedded  material  is  placed,  covering  from  the  air. 
The  chloroform  vapor  hardens  the  mass.  W7hen  it  is  well  set  it  may 
be  transferred  to  a  jar  of  the  chloroform  wrhich  takes  out  the  ether- 
alcohol  and  hardens  the  celloidin  mass,  for  which  a  few^  hours  is 
sufficient.  Allow  the  chloroform  to  act  for  6  to  24  hours.  The 
imbedding  mass  remains  quite  transparent  when  no  water  is  present. 
The  hardening  action  of  the  chloroform  may  be  quickened  and 
intensified  by  carefully  heating  the  chloroform  until  bubbles  of  ether 
begin  to  come  from  the  specimen.  Do  not  let  the  chloroform  evapor- 
ate. 

§  67.  Alcohol  hardening.  When  the  celloidin  mass  is  hard, 
whether  clear  or  not,  it  may  either  be  transferred  to  alcohol  of  about 
82%  strength  in  which  it  is  stored  until  cut,  or  it  may  be  placed  in 
Clarifier  (castor  oil,  1  part;  xylene,  3  or  4  parts).  Alcohol  of  higher 
percentage  softens  the  mass;  lower  grades  such  as  67%  usually  in- 
crease the  hardness  of  the  celloidin  and  in  some  cases  are  to  be 
recommended. 

The  choice  between  alcohol  and  clarifier  involves  no  decision  of  importance 
in  technique.  The  method  of  clarification  has  the  advantage  that  the  orientation 
of  the  specimen  in  the  microtome  preparatory  to  cutting  can  be  more  perfectly 


30 

done.  If  the  tissue  has  been  stained  in  toto  (§  §  79,  142)  the  sections  may  be 
mounted  directly  from  the  clarifier  as  soon  as  cut.  Any  mercuric  chlorid  precipi- 
tate (§11)  that  may  be  present  can  be  dissolved  out  by  means  of  a  solution  of  iodin 
in  the  castor-xylene.  The  castor  oil,  however,  renders  the  microtome,  knife,  hands 
of  the  operator,  etc.,  sticky  and  the  method  is  not  so  cleanly  as  the  alcohol  method. 
On  the  other  hand,  alcohol  tends  to  rust  microtome  and  knife.  Clarification  is 
preferred  by  the  writer  for  serial  work  in  celloidin. 

§  68.  Clarification.  Celloidin  blocks  transferred  from  the 
chloroform  hardener  to  an  oil  mixture  such  as  castor-xylene  (§  67) 
will  become  quite  transparent  (clarified)  and  hardly  discernible,  so 
that  the  tissue  is  readily  seen.  Sometimes,  however,  the  celloidin 
remains  white  and  opaque,  due  to  the  presence  of  moisture,  and 
considerable  time  is  required  for  its  clarification.  In  such  cases  the 
process  may  be  hastened  by  placing  the  tissue  in  the  clarifier  in  a 
warm  place,  and  changing  the  clarifier  several  times.  If  the  block 
still  remains  opaque,  remove  to  95%  alcohol  for  a  day  for  dehydra- 
tion, pass  through  chloroform,  and  into  clarifier.  In  this  way  the 
mass  may  usually  be  cleared  perfectly.  Change  the  clarifier  to  fresh 
after  the  first  day  or  so.  The  sectioning  may  be  done  after  a  few 
hours'  immersion,  although  a  several  days'  clarification  is  preferable.* 

§  69.  Cutting  the  Sections.  There  is  no  marked  difference 
in  the  sectioning  of  celloidin  blocks  preserved  in  alcohol  and  those 
that  have  been  clarified.  In  the  following  paragraphs  67%  alcohol 
should  in  the  reading  be  substituted  for  clarifier  if  alcohol  was  used 
in  the  hardening. 

If  a  paper  box  was  used,  after  the  celloidin  is  ready  for  cutting, 
remove  the  paper,  trim  the  block  as  is  desired  (see  below),  put  some 
thick  celloidin  upon  the  wooden  block  or  other  holder  and  press  the 
base  of  the  celloidin  block  firmly  against  it;  within  two  minutes  it 
will  be  firmly  cemented  and  one  may  proceed  at  once  to  clamp  the 
holder  in  the  microtome  and  commence  cutting. 

For  celloidin  sectioning,  a  long  drawing  cut  is  necessary  in  order 
to  obtain  thin,  perfect  sections.  The  knife  should,  therefore,  be 
set  at  an  obliquity  of  15  to  20°  or  less,  so  that  half  or  more  of  the 


*The  imbedded  object  may  remain  in  the  castor-xylene  clarifier  indefinitely 
without  harm.  The  celloidin  grows  somewhat  tougher  by  a  prolonged  stay  in  it. 
After  cutting  all  the  sections  desired  at  one  time,  the  imbedded  tissue  is  returned 
to  the  clarifier  for  future  sectioning.  It  should  be  remembered,  however,  that 
pure  castor  oil  is  a  solvent  of  celloidin,  hence  it  is  necessary  to  have  the  container 
tightly  stoppered,  otherwise  the  volatile  xylene  will  evaporate  leaving  the  castor 
oil  behind. 


31 

blade  is  used  in  cutting  the  section.  Recall  that  in  the  paraffin 
method  the  knife  is  usually  to  be  set  at  right  angles  to  the  direction  of 
the  cut,  and  the  stroke  is  a  rapid  straight  one.  Trim  away  the  sur- 
rounding celloidin  mass  leaving  enough,  however,  to  serve  as  a  support 
to  the  tissue  and  prevent  its  bending  under  the  impact  of  the  knife ;  if 
the  celloidin  mass  is  too  tapering,  bending  will  occur  and  thin  sections 
cannot  be  cut.  To  avoid  this  the  celloidin  block  is  best  trimmed  in 
the  form  of  a  four  sided  truncated  pyramid  with  as  broad  a  base  as 
possible. 

Clamp  in  the  jaws  of  the  microtome,  placing  it  so  that  the  mass 
of  celloidin  is  opposite  the  side  to  which  the  pressure  of  the  knife  is 
applied  in  cutting.  It  is  advantageous  also  to  have  the  object  placed 
with  its  long  diameter  parallel  with  the  edge  of  the  knife. 

When  knife  and  tissue  are  properly  arranged  wet  the  tissue 
well  with  clarifier  or  alcohol, — as  the  case  may  be, — and  flood  the 
knife  with  the  same.  Make  the  sections  with  a  slow,  steady,  motion 
of  the  knife.  With  a  small  object  (3x5  mm.)  and  a  good  sharp 
knife,  sections  5^  to  6^  can  be  cut  without  difficulty.  In  addition  to 
a  sharp  knife,  however,  there  are  necessary  well-infiltrated  tissue 
and  a  hard,  firm  mass.  If  serial  sections  are  not  desired,  it  may  be 
more  expeditious  to  cut  dry  and  with  a  rapid  stroke. 

§  70.  Transferring  the  sections.  If  the  sections  are  quite 
thick  they  may  be  transferred  from  the  knife  to  a  slide  or  a  dish  by 
means  of  forceps  or  a  brush;  if  they  are  thin,  however,  it  is  better 
to  handle  them  by  means  of  an  absorbent  tissue  paper,  as  follows: 
Flood  the  sections  well  with  clarifier  and  then  by  means  of  a  pipette 
remove  the  clarifier  from  the  knife  and  place  over  the  sections  the 
end  of  a  piece  of  the  tissue  paper,  pressing  it  down  upon  the  sec- 
tions if  necessa^.  Carefully  pull  the  paper  off  the  edge  of  the 
knife;  the  sections  will  adhere  to  the  paper.  Place  the  paper,  sec- 
tions down,  on  a  slide,  taking  care  that  the  sections  are  in  the  de- 
sired position.  With  the  finger  carefully  press  the  sections  (through 
the  transfer  paper)  to  the  slide,  and  then  lift  the  paper,  with  a  roll- 
ing motion,  from  the  slide;  the  sections  will  adhere  to  the  slide. 
Should  they  stick  to  the  paper  instead,  lower  the  paper  again  and 
again  firmly  press  the  section  to  the  slide.  For  further  procedure 
see  §  §  135,  136.  If  it  is  not  desired  to  mount  the  sections  upon  a 
slide  immediately,  or  if  they  are  to  be  kept  in  bulk,  as  for  class 
work,  the  transfer  paper  may  be  shaken  gently  in  a  dish  of  clarifier 


32 

or  05%  alcohol  and  the  section  (or  sections)  will  float  free  and  sink 
to  the  bottom. 

§  71.  Serial  Sectioning.  If  it  is  desired  to  mount  the  sections 
in  series,  proceed  as  follows:  With  a. camel's  hair  brush  or  needle 
draw  the  first  section,  when  cut,  up  toward  the  back  of  the  knife  and 
make  the  next  section.  Place  this  section  to  the  right  of  the  first,  and 
so  on,  arranging  them  in  serial  order,  section  after  section,  and  line 
below  line,  until  enough  are  cut  to  fill  the  area  that  the  cover-glass  will 
cover.  Flood  the  sections  as  before  by  letting  the  clarifier  flow  over 
them,  being  careful,  however,  not  to  float  them  from  their  places. 
Absorb  the  clarifier  from  the  knife  with  a  pipette,  and  place  over 
the  sections  a  piece  of  the  transfer  paper  twice  the  width  of  a  slide; 
press  it  down  if  necessary,  and  slowly  draw  it  off  the  edge  of  the 
knife.  Should  it  then  be  seen  that  some  of  the  sections  are  adhering 
to  the  knife  instead  of  the  paper,  it  means  that  the  clarifier  had  been 
allowed  to  thicken*  on  them,  cementing  them  to  the  knife,  and  the 
preliminary  flooding  to  insure  their  being  free,  was  insufficient.  In 
that  case  it  is  best  to  flood  the  paper  with  clarifier,  carefully  lift  it, 
arrange  the  sections  again,  flood  them  with  clarifier,  place  a  clean 
piece  of  transfer  paper  over  them  and  try  again.  One  soon  becomes 
accustomed  to  the  behavior  of  the  sections,  and  accidents  are  rare. 
In  cutting  a  series  of  many  small  sections,  some  time  is  consumed 
and  it  is  necessary  to  flood  the  sections  on  the  knife  frequently  with 
clarifier  while  cutting  in  order  to  prevent  the  clarifier  thickening  and 
cementing  them  to  the  knife. 

§  72.  Resume  of  the  method.  Success  in  the  employment  of 
the  celloidin  or  collodion  method  depends  upon  the  thorough  infil- 
tration with  the  solutions,  requiring  days  or  even  months,  and  the 
employment  of  a  thick  imbedding  mass  giving  when  hardened  a  firm 
unyielding  support  to  the  tissue.  This  may  be  gained  by  employing 
a  relatively  long  period  of  infiltration,  and  taking  pains  in  imbed- 
ding to  have  the  imbedding  mass  well  thickened. 

Observing  these  two  cautions,  celloidin  may  be  used  in  almost 
all  cases  as  an  imbedding  mass,  except  such  as  are  affected  by  the 
conditions  of  the  methods  already  mentioned  (§  §  47  and  61). 


*If  one  is  a  long  time  cutting  a  series  of  sections,  it  sometimes  occurs  that 
the  xylene  evaporates  leaving  the  castor  oil  that  is  thick  and  viscid  and  also 
a  solvent  of  the  collodion,  so  that  the  sections  are  not  easily  transferable  but  stick 
rather  firmly  to  the  knife.  In  such  a  case,  fresh  clarifier  or  even  a  little  xylene 
to  dissolve  the  castor  oil  must  be  used. 


33 

THE    FREEZING    METHOD. 

§  73.  This  method  is  expeditious  and  of  use  in  the  rapid  ex- 
amination of  tissues,  and  therefore  especially  serviceable  in  the 
pathological  laboratory  and  in  clinical  diagnoses.  It  may  also  be 
used  in  cutting  tissues  that  are  too  hard  to  be  cut  satisfactorily  by 
means  of  either  the  collodion  or  paraffin  methods,  and  in  the  exami- 
nation of  tissues  for  substances  (e.  g.,  fats)  which  the  solutions 
necessary  for  the  paraffin  and  celloidin  methods  dissolve  out.  Both 
fresh  and  fixed  tissue  may  be  cut  by  means  of  the  freezing  micro- 
tome and  with  or  without  any  special  mass  such  as  is  used  in  paraffin 
or  celloidin  imbedding.  Some  histologists  quite  prefer  the  freezing 
method  to  the  paraffin  or  celloidin  methods  for  general  use. 

When  no  mass  is  employed  the  tissue  is  simply  frozen  and  cut, 
or,  if  it  is  fixed  tissue,  soaked  well  in  water  first  and  then  frozen. 
When  extreme  haste  is  not  so  essential  it  is  better  to  first  saturate 
the  tissue  with  some  solution  that  does  not  crystallize  on  freezing, 
but  simply  hardens,  since  the  formation  of  the  ice  crystals  is  hurtful 
to  the  tissue.  Such  are  solutions  of  gum  arable  or  sugar  and  anise- 
seed  oil,  and  they  are  spoken  of  as  Congelation  masses. 

§  74.  Infiltration.  Gum  arabic  or  anise-seed  oil  may  be  used. 
(a)  Gum  arabic.  If  the  tissue  has  been  fixed  and  is  in  alcohol  re- 
move the  alcohol  by  soaking  it  for  several  hours  to  1  day  in  water. 
Remove  to  a  thick  solution  of  gum  arabic  in  water,  in  which  it  may 
remain  for  about  24  hours.  It  is  then  ready  ta  freeze  and  cut. 

(b)  Anise-seed  oil.  For  this  method  the  tissue  should  be  first 
dehydrated  (§  51).*  W^hen  dehydration  is  complete,  transfer  the 
tissue  to  anise-seed  oil,  in  which  it  may  soak  for  12  to  24  hours;  it  is 
then  ready  to  freeze  and  cut.  It  is  particularly  adapted  for  use  with 
tissue  that  has  been  stained  in  toto. 

§  75.  Cutting.  Place  a  drop  of  the  solution  of  gum  arabic 
(or  anise-seed  oil)  upon  the  object  carrier  of  the  freezing  microtome 
and  turn  on  the  carbonic  acid  (or  ether)  spray.  When  the  mixture 
begins  to  harden,  place  the  object  upon  it  in  an  abundance  of  the 
solution  and  freeze  it  nearly  solid.  Covering  with  an  inverted  cup 
hastens  the  freezing.  An  especially  wedge-shaped  knife  is  necessary 
because  of  the  hardness  of  the  mass. 


*Anise-seed  oil  will,  however,  clear  from  90%  or  even  82%  alcohol;    this 
is  sometimes  of  advantage. 


34 

When  the  tissue  is  completely  frozen,  cut  it  with  a  straight  move- 
ment of  the  knife,  as  in  the  paraffin  method,  holding  it  firmly  upon  the 
knife  rest  and  making  the  strokes  as  rapidly  as  possible,  at  the  same 
time  rapidly  raising  the  tissue  a  few  microns  at  a  time  by  means  of 
the  microtome  screw.  There  are  a  number  of  automatic  microtomes 
specially  designed  for  use  \vith  the  freezing  method. 

The  mass  of  sections  is  transferred  to  a  dish  of  water  in  which 
the  gum  arabic  is  dissolved  away  and  the  sections  are  ready  for  stain- 
ing (§  §  137,  146).  If  anise-seed  oil  is  used,  the  sections  are  to  be 
transferred  to  95%  alcohol  which  will  dissolve  out  the  oil;  if  the 
tissue  has  been  stained  in  toto  the  sections  may  be  transferred  to 
anise-seed  oil  (or  other  clearer)  and  mounted  in  balsam  directly. 

§  76.  Rapid  Method.  Blocks  of  tissue  1  centimeter  thick  should 
fix  in  10%  formalin  12  to  24  hours.  If  haste  is  a  factor,  take  thinner 
pieces  and  fix  for  1  minute  or  more.  Trim  the  block  so  that  it  is  about 
5  mm.  thick;  rinse  in  water  for  a  few  seconds,  transfer  to  the  freezing 
microtome,  freeze  and  section. 

Float  the  sections  when  cut  from  the  knife  into  water  from  which 
they  may  be  gotten  upon  the  slides  by  means  of  a  camel's  hair 
brush.  Drain  off  the  water  and  press  the  sections  out  smooth  by 
means  of  blotting  paper,  filter  paper  or  other  absorbent  paper.  Cau- 
tiously drop  over  the  sections  95%  and  absolute  alcohol  and  follow 
this  immediately  with  thin  (%  or  /4%)  celloidin  solution  (§  64) 
which  when  it  has  partially  evaporated  out  will  serve  to  support  the 
section  and-  fasten  it  to  the  slide.  It  is  now  ready  for  staining. 
(§  144).  

STAINING. 

§  77.  Staining  has  for  its  first  and  primary  purpose,  the  ren- 
dering outlines  and  structures  more  distinct  by  giving  them  a  color 
contrast  with  their  surroundings  (color  image).  A  second  and 
more  important  use  is  for  the  differentiation  of  particular  struc- 
tures or  substances  which  by  their  selective  staining  facilitate  the 
histological  analysis.  Rational  staining,  like  rational  fixation,  de- 
pends upon  the  physics  and  chemistry  of  staining  reactions;  indeed, 
in  the  demonstration  of  particular  substances  the  fixation  and  stain- 
ing should  be  determined  by  the  mutual  interdependence  of  their  re- 
actions, since  they  have  the  same  purpose, — the  preservation  and 
demonstration  of  the  substance  sought  for. 


35 

In  some  cases  the  differential  staining  may  be  accomplished  in 
the  fixing  (fats,  impregnations).  Differential  staining,  histo-chemical 
methods,  micro-chemical  methods  (as  applied  to  the  animal  organism) 
thus  belong  in  the  same  category  and  rest  upon  a  physico-chemical 
basis.  As  in  the  case  of  fixation,  a  great  deal  remains  to  be  done 
in  the  perfection  of  this  side  of  histological  technique. 

§  78.  Classification  of  Stains.  Stains  may  be  grouped:  (a) 
according  to  their  chemical  composition  as  (1)  organic; — hematoxylin 
stains,  carmine  stains,  anilin  stains,  (coal-tar  dyes;  benzene  deriva- 
tives), and  (2)  inorganic,  (b)  From  another  chemical  aspect  as  (1) 
basic  or  (2)  acid,  depending  upon  the  chemical  reaction  of  the  staining 
principle  or  group,  (3)  neutral  (§  85).  (c)  Histologically,  stains  are: 
(1)  nuclear  (chromatin  stains),  (2)  plasma  or  general  stains,  (3)  special 
stains,  (4)  impregnations.  The  first  are  usually  basic,  the  second 
acid,  the  fourth  inorganic. 

§  79.  In  toto  staining.  When  in  toto  or  bulk  staining  is  em- 
ployed, the  piece  of  tissue  is  stained  entire  and  imbedded  and  sec- 
tioned afterwards.  In  this  case  the  tissue  should  be  stained  before 
the  process  of  embedding  has  begun,  after  the  washing  out  of  the 
fixer  has  been  completed  (§  §  7,  9).  But  a  single  stain  may  be  given 
and  the  one  chosen  is  generally  a  nuclear  one, — hematoxylin,  cochi- 
neal, or  carmine  (§  §  91,  98 — ).  A  counter  stain  may  be  given  sub- 
sequently after  sectioning,  orange  G  after  hematoxylin  or  cochineal, 
Lyon's  blue  after  carmine  being  most  satisfactory.  In  toto  staining 
is  particularly  useful  in  serial  sections  of  embryological  material  and 
in  morphological  work  in  general,  as  it  saves  time  and  manipulation. 

§  80.  Section  Staining.  The  application  of  the  staining  proc- 
esses after  the  tissue  is  imbedded  and  sectioned.  This  is  more 
serviceable,  especially  if  highly  differential  results  are  desired. 

§  81.  Progressive  and  Regressive  Staining.  According  to  the 
mode  of  application,  staining  is  either  progressive  or  regressive. 
Progressive  staining  consists  in  permitting  the  staining  to  proceed 
to  the  right  degree  of  intensity  and  then  stopping  it.  In  regressive 
staining  the  tissue  is  over-stained  and  the  excess  of  stain  removed 
by  the  application  of  a  Differentiator.  Uusally  in  the  regressive 
method  there  is  employed  a  Mordant  which  is  a  chemical  solution 
serving  to  make  the  stain  "take."  It  possesses  a  double  affinity, — 
to  the  tissue  and  to  the  stain,  which  is  usually  made  to  operate  in  the 
interest  of  differential  or  selective  staining.  The  mordant  is  not 


36 

always  in  the  form  of  a  separate  solution  (e.  g.,  aluminium  hema- 
toxylin)  and  in  many  cases  the  mordantage  is  given  in  the  fixing. 
Mordantage,  directly  or  indirectly  employed,  is  also  useful  in  staining 
by  the  progressive  method.  Stains  or  dyes  that  do  not  require  a 
mordant  are  termed  Substantive,  those  requiring  a  mordant  to  make 
them  "take"  are  Adjective  stains. 

Delicate  results  in  exact  differential  staining  by  either  the  progres- 
sive or  regressive  methods  can  best  be  secured  only  by  section  stain- 
ing, although  differentiation  of  the  sections  after  in  toto  staining  may 
be  resorted  to. 

§  82.  Differentiation.  In  the  regressive  method  it  is  neces- 
sary to  remove  the  excess  of  stain  by  the  application  of  a  solution 
that  will  usually  differentiate  it,  or  bring  out  the  selective  action  of 
the  stain.  A  small  amount  of  differentiation  is  usually  necessary 
in  any  case  for  the  most  delicate  results.  Alcohol,  95%  or  other 
grade,  may  often  be  used  as  a  differentiator.  95%  acidified  with 
hydrochloric  acid  (1/10  to  1%)  is  sometimes  used,  or^a  special  dif- 
ferentiator is  required  (acetone,  alum  solution,  clove-oil,  etc.). 
It  is  usually  necessary  to  control  the  differentiation  by  use  of  the 
microscope. 

§  83.  Impregnations.  In  addition  to  the  typical  methods  of 
coloring  tissue  by  means  of  stains  there  is  a  group  of  methods  in 
which  the  coloring  matter  is  deposited  in  the  cell  or  tissue  that  it  is 
desired  to  differentiate,  in  the  form  of  a  precipitate.  These  are 
known  as  impregnation  methods  and  are  of  great  value,  especially 
as  applied  to  nervous  tissues.  A  hard  and  fast  line,  however,  can- 
not be  drawn  between  true  staining  methods  and  impregnations. 
Silver  nitrate  and  gold  chlorid  are  the  substances  most  generally 
employed  in  the  impregnation  of  tissue  (§  §  221,  223). 

§  84.  Choice  of  stains.  Remember  that  the  staining  is  greatly 
affected  by  the  previous  treatment,  brilliancy  or  selectivity  in  the 
result  being  in  many  cases  dependent  on  the  fixer  employed  or  im- 
paired by  the  improper  or  incomplete  washing  out  of  the  fixer. 

In  staining,  therefore,  consider  three  things,  (a)  what  it  is  de- 
sired to  bring  out, — what  kinds  of  stains  you  need  to  employ,  (b) 
the  mode  of  fixation  that  has  been  emploj^ed,  and  (c)  the  imbedding 
method  must  also  be  considered  in  the  choice,  since  if  celloidin  is 
employed  certain  stains  that  color  it  deeply  should  be  avoided. 


37 

(a)  For  general  purposes  it  is  customary  to  use  a  nuclear  and 
a   cytoplasmic   stain   either  in   combination   or  successively.     The 
double  staining  most  employed  is  hematoxylin  and  eosin.     Triple 
stains  are  generally  not  so  satisfactory;    hematoxylin  and  picro- 
fuchsin  is  one  commonly  used.     Neutral  stains  (§  85)  are  essentially 
triple  stains.     Quadruple  stains  are  rarely  serviceable;   see,  however, 
§  129.     For  stains  to  be  employed  for  special  purposes,  see  Special 
Methods,  §  174-. 

(b)  In  the  case  of  most  of  the  fixers  given  §  11-,  there  are  no 
restrictions  as  to  the  stains  that  may  be  employed  though  some 
are   more   satisfactory   than   others.     With   Flemming's   fluid   and 
Hermann's  fluid,  however,  it  is  well  to  employ  only  such  stains 
as  iron  hematoxylin  (§94)  and  anilin  stains, — safranin  being  suggested 
as  a  red  stain,  gentian  violet  as  a  blue  stain.     Dichromate  fixers  whose 
action  has  been  prolonged  usually  require  strong  stains.     Iron  hema- 
toxylin and  similar  stains  may  be  used  after  any  fixer.     The  Ehrlich- 
Biondi-Haidenhain  stain  (§  111)  is  most  satisfactory  after  Mercuric 
chlorid  (§  11). 

(c)  Celloidin  is  deeply  stained  by  such  stains  as  iron  hema- 
toxylin, safranin,  methylene  blue,  gentian  violet,  and  basic  stains  in 
general.     If  it  is  necessary  to  use  these,  with  celloidin  material,  the 
celloidin   should   be   removed   from   the   sections   before   mounting 
(§   136). 

§  85.  Neutral  Stains.  In  the  case  of  most  of  the  anilin  stains,  if  an  aqueous 
solution  of  a  basic  stain  is  added  to  an  aqueous  solution  of  an  acid  stain,  there  is 
formed  by  combination  a  neutral  stain  which  is  usually  but  slightly  soluble  in 
water  and  hence  precipitates  out.  Neutral  stains  are  however  moderately  soluble 
in  strong  methyl  or  ethyl  alcohol.  These  facts  it  is  well  to  keep  in  mind  in  using 
anilin  stains.  The  reaction  may  be  made  use  of  for  increasing  the  selectivity, 
sharpness  and  color  tones  of  the  original  stains.  Neutral  stains  may  be  used  in 
one  of  three  ways:  (a)  The  neutral  stain  is  often  soluble  in  excess  of  the  acid 
or  basic  stain  or  in  an  aqueous  solution  of  another  acid  or  basic  stain  (§111).  (b) 
Dissolved  in  methyl  (or  ethyl)  alcohol  and  diluted  nearly  to  the  precipitation  point 
the  activity  and  selectivity  of  the  component  stains  seem  to  become  specially 
marked  (§  128,  214).  (c)  The  formation  of  the  neutral  stain  may  be  carried  out 
in  the  staining  process  itself, — "on  the  slide," — by  staining  first  strongly  with  an 
aqueous  solution  of  the  acid  stain,  rinsing  away  the  excess  stain  and  staining  with 
the  aqueous  solution  of  the  basic  stain  (§  106)  and  differentiating  (§  82)  with 
alcohol,  or  alcohol  and  clove  oil. 

Of  the  large  number  of  stains  that  combine  to  forrn  neutral  stains  may  be 
mentioned, — the  acid  stain  being  given  first; — eosin  and  methylene  blue  (§  128); 
orange  G.  and  gentian  violet  (§  131),  erythrosin  and  toluidin  blue,  thiazin  red  and 
toluidin  blue,  coerulein  S.  and  safranin,  acid  violet  and  safranin.  These  may  be 
combined  and  used  in  any  one  of  the  three  ways  mentioned  above. 


38 

§  86.  The  time  of  staining.  Although  in  general  certain  time 
limits  can  be  given  to  the  period  during  which  a  stain  should  be 
allowed  to  act,  with  most  stains,  especially  those  with  which  no  dif- 
ferentiation is  needed,  such  as  hematoxylin,  and  most  carmines,  the 
correct  intensity  of  color  should  be  determined  by  examining  the 
preparation  with  the  microscope.  One  soon  becomes  able  to  judge 
of  the  right  stain  in  this  way  better  than  if  a  given  time  were  adhered 
to. 

In  the  use  of  stains  requiring  a  subsequent  differentiation,  the 
rule  is  'to  over-stain  and  watch  the  differentiation  carefully  with  the 
microscope,  stopping  it  when  sufficient.  In  this  case  it  is  the  differ- 
entiation and  not  the  staining  that  should  be  carefully  regulated. 
In  general,  for  the  best  results,  it  is  advisable  to  use  staining  and 
differentiating  solutions  in  dilute  form  and  prolong  the  time  during 
which  they  act. 

The  following  formulas  include  the  more  generally  useful  stains 
and  those  to  be  employed  in  the  "Special  Methods"  given  subse- 
quently. 

STAINS. 

§  87.  Hematoxylin  Stains.  Hematoxylin  is  a  colorless  compound  of  acid 
properties  forming  therefore  salts  with  bases  which  oxidize  readily  forming 
"hemateates."  The  oxidation  product  of  hematoxylin  is  hematein  which  is 
the  real  staining  principle  and  may  in  some  cases  be  used  with  real  advantage 
instead  of  hematoxylin.  Hematoxylin  (hematein)  itself  has  little  value  as  an 
acid  (plasma)  stain;  combined  with  metallic  bases  it  becomes  a  valuable  basic 
(chromatin)  stain.  The  metals  usually  employed  as  mordants  for  hematoxylin 
are:  aluminium,  iron,  copper,  chromium,  molybdenum,  vanadium.  Their 
salts  may  be  used  either  in  the  same  solution  as  the  hematoxylin  (aluminium, 
molybdenum,  vanadium)  or  separately  (iron,  copper.)  Solutions  of  the  same 
metals  may  in  some  instances  (aluminium,  iron,  chromium,)  be  employed  also  as 
differentiator.  For  preparing  hematoxylin  stains  it  is  a  great  convenience  to 
have  a  10%  stock  solution  in  95%  alcohol. 

§  88.  Chloral  Hematoxylin.  [11]  Formula:  Potassium  alum,  8  grams; 
distilled  water,  250  c.  c.;  hematoxylin  2/10th  gram  or  2  c.  c.  hematoxylin  stock 
solution.  Boil  5  or  10  minutes  in  an  agate  dish.  After  cooling  add  6  grams  of 
chloral  hydrate.  Place  in  a  bottle  and  permit  the  hematoxylin  to  oxidize  for  a 
week  or  two,  or  1  to  2  c.  c.  hydrogen  peroxid  may  be  added.  Its  staining  quality 
improves  up  to  an  optimum  and  then  begins  to  deteriorate.  Old  hematoxylin 
generally  contains  a  precipitate  and  should  be  filtered  often  or  before  using. 

Stain  sections  5  to  30  minutes  according  to  the  age  of  the  solution,  the  charac- 
ter of  the  tissue  and  the  fixation  employed.  After  staining  wash  well  with  dis- 
tilled or  tap  water.  Usually  no  differentiation  is  required  unless  a  purer  chroma- 
tin  stain  is  desired  (§  175).  Counter  stain  as  desired. 


39 

§  89.  Mayer's  Haemalum.  Formula:  haematein,  1  gram;  90%  (95%) 
alcohol,  50  c.  c.;  potash  alum,  50  grams;  distilled  water,  1,000  c.  c.  Dissolve 
the  haematein  in  the  alcohol,  the  alum  in  the  water;  mix. 

This  is  an  excellent  formula,  giving  a  good  stain  immediately  after  it  is  made 
up  and  retaining  its  selective  staining  quality  for  a  year  or  longer.  It  is  one  of  the 
best  hematoxylins  for  ordinary  work. 

For  this  formula,  Mayer  has  now  substituted  the  following:  hematoxylin, 
1  gram  (10  cc.  stock  solution);  distilled  water,  1,000  c.  c.;  sodium  iodate,  0.2 
grams;  potassium  alum,  50  grams.  The  hematoxylin  is  first  dissolved  in  the 
water  and  then  the  other  ingredients  are  added.  Dissolve  and  filter.  50  grams 
of  chloral  hydrate  and  1  gram  of  citric  or  acetic  acid  may  be  added  as  a  preserva- 
tive. Stain  as  given  in  §  88.  In  using  it  is  frequently  advisable  to  dilute  one  or 
more  times  with  distilled  \vater. 

§  90.  Ehrlich's  acid  hematoxylin.  Formula:  Water,  100  c.  c.;  95% 
alcohol,  100  c.  c.  hematoxylin  crystals,  2  grams  (20  c.  c.  stock  solution) ;  dissolve  in 
the  alcohol;  glycerin,  100  c.  c.;  glacial  acetic  acid,  10  c.  c.;  alum  in  excess.  Let 
the  mixture  ripen  in  the  light  until  a  dark  red.  Sections  stain  in  this  hematoxylin 
in  a  short  time,  generally  5  to  10  minutes.  Wash  with  water  after  staining. 

§  91.  Delafield's  hematoxylin.  Formula:  Saturated  aqueous  solution 
of  ammonia  alum,  200  c.  c.;  hematoxylin  .stock  solution  20  c.  c.  Allow  the 
mixture  to  stand  in  the  light  and  air  in  an  unstoppered  bottle  for  4  or  5  days; 
filter  and  add  glycerin,  50  c.  c.,  and  methyl  alcohol,  50  c.  c.  Permit  it  to  stand 
for  a  week  or  so  to  ripen;  filter  and  keep  in  a  stoppered  bottle.  The  staining 
power  increases  for  several  months.  In  using,  dilute  3  or  4  times  or  more  with 
distilled  water.  It  is  useful  for  in  toto  staining  diluted  1 :9  with  20%  alcohol  or 
distilled  water. 

Stain  sections  from  water;  4  to  5  minutes  will  generally  be  sufficient.  Wash 
well  with  water  after  staining.  In  toto  staining  1  to  14  days  may  be  necessary 
depending  upon  the  size  of  the  object. 

This  is  a  very  strong  hematoxylin  stain  and  may  be  used  to  advantage  with 
tissues  that  stain  with  difficulty.  It  is  likewise  a  more  diffuse  stain  than  either 
chloral  or  Ehrlich's  hematoxylin,  staining  cell-body  as  well  as  nucleus, — a  feature 
having  its  advantages.  Old  solutions  (several  months  to  a  year)  should  be 
filtered  before  using. 

§  92.  Acid  Hematoxylin  (Delafield's).  The  dilution  of  Delafield's  hema- 
toxylin ten  to  twenty  times  with  water,  and  making  slightly  acid  with  acetic 
gives  a  useful  stain  particularly  for  differentiating  nuclei  (embryological  work.) 

§  93.  Muchematein  (Mayer).  Aqeuous  formula:  Rub  up  0.2  grm.  hema- 
tein  with  a  few  drops  of  glycerin;  add  0.1  grm.  aluminium  chlorid,  40  c.  c.  glycerin, 
60  c.  c.  distilled  water.  Filter  if  necessary.  Alcoholic  formula:  Hematein,  0.2 
grms.;  aluminium  chlorid,  0.1  grm.;  70%  (67%)  alcohol,  100  c.  c.;  1  or  2  drops 
nitric  acid.  Formulae  with  a  minimum  amount  of  aluminium  designed  as  specific 
stain  for  mucus.  The  aqueous  solution  is  more  selective;  the  alcoholic  formula 
designed  for  the  staining  of  mucus  that  swells  excessively  in  water  (§  234). 

§  94.     Iron     Hematoxylin     (Heidenhain).     Formula:     (a)     Mordant-      2% 
aqueous    solution    of    ferric    alum    (iron-ammonium-persulphate),     (b)    Stain; 
aqueous  solution  of  hematoxylin   (10%  alcoholic  stock  solution,  5  c.  c.; 


40 

distilled  water,  95  c.  c.).  (c)  Differentiator;  the  ferric  alum  mordant,  preferably 
diluted  several  times.  An  excellent  stain,  especially  for  cytological  work.  It 
may  be  used  after  any  fixer. 

The  steps  necessary  are:  (1)  Mordanting  1  to  24  hours;  (2)  rinse  the,  sec- 
tions in  water  10  to  30  minutes;  (3)  stain  for  3  to  24  hours;  differentiate  slowly 
and  control  it  under  the  microscope.  The  slides  may  be  alternately  dipped  into 
the  ferric  alum  solution  for  a  few  seconds  and  then  into  tap  water.  (4)  Wash 
in  running  water  15  to  60  minutes.  The  ferric  alum  mordant  may  be  used  several 
times  as  well  as  the  hematoxylin  solution  whose  staining  quality  improves  up  to 
a  limit  by  use. 

§  95.  Copper  Hematoxylin  (Weigert).  Formula:  Mordant;  3.5%  aqueous 
solution  of  copper  acetate;  Stain;  10%  alcoholic  solution  of  hematoxylin  10  c.  c.; 
distilled  water,  90  c.  c.;  saturated  solution  lithium  carbonate,  1  c.  c.  Differentia- 
tor; potassium  ferricyanide,  2.5  grms.,  borax,  2  grms.;  distilled  water,  200  c.  c. 
Designed  for  staining  the  myelinic  sheath  of  medullated  nerve  fibers  (§  199)  but 
useful  for  other  purposes. 

The  steps  are:  (1)  mordanting  for  1  to  24  hours,  (2)  rinsing  the  sections 
in  water  10  to  30  minutes,  (3)  staining  for  3  to  24  hours,  (4)  differentiate  slowly 
controlling  the  action  under  the  microscope.  (5)  Wash  in  running  water  30 
minutes  or  longer.  The  mordantage  may  be  given  in  bulk  if  desired.  As  usually 
employed  with  tissue  fixed  in  chrome  fixers,  the  stain  is  strictly  a  chrome-copper 
hematoxylin.  Other  differentiators  may  be  used  if  desired.  Mount  in  neutral 
balsam. 

§  96.  For  Mallory's  phospho-molybdic  hematoxylin,  Heidenhain's  vanadium 
hematoxylin,  and  other  hematoxylin  formulae  of  occasional  usefulness,  consult 
the  works  on  technique  (p.  90). 

§  97.  Carmine  Stains'.  Like  hematoxylin,  the  carmine  stains  depend 
upon  an  acid  staining  principle, — carminic  acid, — which  in  combination  with 
bases  gives  a  red  nuclear  stain  of  value.  The  metals  usually  employed  in  carmine 
formulae  are  aluminium,  calcium,  strontium,  iron.  Carmine  itself  is  (Liebermann) 
a  combination  of  carminic  acid  with  aluminium,  calcium,  and  protein.  It  is 
soluble  in  acids  and  alkalis.  The  necessary  mordant  is  not  so  often  employed 
in  a  separate  solution  as  is  the  case  with  hematoxylin.  See,  however,  iron  carmine 
[6,  30]. 

§  98.  Carmalum  (Mayer's)  Formula:  Carminic  acid,  1  grm.;  potas- 
sium alum,  10  grms.;  distilled  water,  200  c.  c.  Dissolve  with  heat  (if  neces- 
sary). Filter.  Add  1  c.  c.  formalin  as  a  preservative.  It  may  be  used  for 
in  toto  or  for  section  staining. 

Stain  sections  5  to  30  minutes  or  as  long  as  necessary.  Rinse  tissue  before 
staining  with  distilled  (not  tap-)  water. 

§99.  Borax  carmine.  (Grenacher).  Formula:  Borax  4  grams;' carmine,  3 
grams;  water,  100  c.  c.;  allow  the  mixture  to  stand  for  several  days,  shaking 
occasionally  when  most  of  the  carmine  will  have  dissolved;  filter  and  add  100 
c.  c.  of  70%  alcohol.  Let  the  mixture  remain  for  several  days,  filter  again  and 
the  solution  is  ready  for  use. 

This  is  a  good  carmine  stain  for  in  toto  staining.  Stain  objects  in  toto  for  one 
to  several  days,  according  to  size;  remove  to  67%  (70%)  alcohol,  acidulated 


41 

slightly  with  hydrochloric  acid,  (4  drops  in  each  100  c.  c.),  for  a  day  and  then 
remove  to  80%  alcohol.     It  affords  a  bright  red  stain  that  is  quite  transparent. 

§  100.  Paracarmine  (Mayer).  Formula:  Carminic  acid,  1  gram;  alumi- 
nium chlorid,  0.5  gram;  calcium  chlorid,  4  grams;  70%  (67%)  alcohol,  100  c.  c. 
Allow  it  to  stand  a  day  or  so,  shaking  occasionally  until  the  carminic  acid  has 
quite  dissolved,  and  then  filter. 

This  is  an  excellent  carmine  stain  for  in  toto  staining.  The  tissue  may  be 
stained  one  to  several  days  (1  week),  then  washed  in  67%  and  82%  alcohols  to 
remove  the  excess  of  staining  fluid.  A  red  nuclear  stain,  more  opaque  than  borax 
carmine.  It  does  not  over-stain  readily,  and  since  it  is  an  alcoholic  solution  (70%) 
it  is  quite  penetrating  and  may  be  allowed  to  act  for  a  greater  length  of  time, 
being  thus  suited  for  staining  in  toto  objects  of  considerable  size. 

§  101.  Hcl.  carmine.  Formula:  Carmine,  2  grams;  concentrated  hydro- 
chloric acid,  3  c.  c.;  70%  alcohol,  100  c.  c.  Boil  gently  for  15  to  20  minutes  to 
dissolve  the  carmine;  cool  and  filter. 

This  is  a  strong  carmine  stain,  quite  suitable  for  sections,  especially  such  as 
stain  with  difficulty.  It  may  also  be  employed  for  staining  in  toto.  Stain  sec- 
tions from  alcohol  or  water  for  5  to  15  minutes ;  rinse  away  the  superfluous  stain 
with  67%  (70%)  alcohol  and  differentiate  for  a  few  seconds  to  a  minute  with  acid 
alcohol  (95%  alcohol  100  c.  c.,  concentrated  hydrochloric  acid  1/10  c.  c.).  Wash 
away  the  acid  alcohol  with  ordinary  95%  alcohol.  If  a  pure  nuclear  stain  is  not 
desired  the  differentiation  may  be  omitted. 

Picric  acid  (§  116)  may  be  used  as  a  counter  stain,  and  in  that  case  differentia- 
tion is  ordinarily  not  required. 

§  102.  Alum  Cochineal.  Formula:  Powdered  cochineal,  75  grams;  potas- 
sium alum,  75  grams;  distilled  water,  1000  c.  c.  Boil  the  ingredients  for  half  an 
hour,  or  (better)  macerate  for  a  day  or  so,  boiling  up  two  or  three  times.  Cool 
and  filter.  Add  to  the  filtrate  distilled  water  to  make  up  1000  c.  c.  and  a  crystal 
of  thymol  as  a  preservative. 

This  is  an  excellent  stain,  particularly  for  in  toto  staining  of  embryos,  giving  a 
purpler  stain  than  the  carmine  stains  given  above. 

Stain  objects,  such  as  embryos,  over  night  to  2  days  or  longer,  depending  on 
size.  Wash  out  with  water,  2  to  6  hours  and  place  successively  in  50%,  67%  and 
82%  alcohols  several  hours  to  a  day  in  each. 

§  103.  Mucicarmine.  Formula:  Carmine,  1.  gram;  alummium  chlorid 
(pure),  0.5  grams;  distilled  water,  2  c.  c.  Mix  thoroughly  together  and  heat  over 
a  small  flame  for  2  minutes  (in  a  test  tube)  until  the  mixture  has  become  dark  red. 
Dissolve  the  whole  in  100  c.  c.  50%  alcohol  added  gradually;  after  24  hours 
filter.  For  use,  it  may  be  diluted  5  or  10  times  with  water. 

Stain  sections  10  minutes  or  longer  in  the  diluted  stain,  rinse  in  water,  dehy- 
drate, clear,  and  mount  in  neutral  balsam. 

If  desired,  the  stain  may  be  diluted  with  50  or  67%  alcohol  instead  of  water. 

§  104.  Gentian  Violet.  Formula:  A  concentrated  solution  in  distilled 
water.  Stain  (paraffin)  sections  from  water  for  5  to  10  minutes,  rinse  in  water, 
dehydrate  and  differentiate  with  95%  alcohol  and  complete  the  differentiation 
with  clove  oil.  When  the  differentiation  is  sufficient,  clear  with  bergamot  oil 


42 

and  mount  in  balsam.  This  may  be  used  alone  to  give  a  blue  stain  with  tissue 
fixed  in  Hermann's  or  Flemming's  fluid.  (See  also  §  112,  131). 

§  105.  Methylene  Blue.  This  valuable  stain,  used  particularly  in  the 
histology  and  pathology  of  the  blood  and  nervous  system,  and  in  bacteriology, 
is  represented  in  a  large  number  of  formulae.  For  the  staining  of  nuclei,  basic 
granules  in  the  cytoplasm,  neurochromatin  granules,  etc.,  simple  aqueous  solu- 
tions may  be  employed.  A  1%  solution  suffices  for  most  purposes;  in  some 
cases,  a  concentrated  solution  is  to  be  preferred. 

Stain  from  water  for  5  minutes  to  as  many  hours,  with  or  without  heat,  rinse 
with  distilled  water,  differentiate  if  desired  in  a  l/10th%  Hcl.  in  95%  alcohol, 
or  l/10th%  alum  solution.  Wash,  dehydrate,  mount  in  neutral  balsam. 

§  106.  Alkaline  Methylene  Blue.  Formula:  Methylene  blue,  2  grms.; 
absolute  or  95%  alcohol  neutralized  with  pure  dry  sodium  carbonate,  50  c.  c.; 
add  distilled  water,  450  c.  c.;  1%  potassium  hydroxid,  5  c.  c.  An  excellent  stain, 
giving  best  results  after  mercuric  chlorid  fixers  (incl.  Zenker's,  etc.). 

§  107.  Eosin— Methylene  Blue.  Stain  sections  l/2  hour  with  a  l/4%  to  1% 
aqueous  solution  of  eosin,  rinse  in  water,  stain  in  alkaline  methylene  blue  10 
minutes,  rinse  well  in  water.  Differentiate  and  dehydrate  rapidly  with  neutral 
95%  alcohol  and  absolute  alcohol,  clear  in  xylene,  mount  in  neutral  balsam. 
Particularly  useful  for  staining  blood  in  the  tissues  (hemolymph)  glands,  etc.. 

§  108.  Toluidin  Blue.  This  may  be  used,  often  to  advantage,  in  place  of 
and  for  the  same  purposes  as  methylene  blue.  It  gives  a  somewhat  darker  stain. 

§  109.  Methyl  green.  This  is  a  nuclear  stain  of  much  value,  besides  being 
an  important  ingredient  of  triple  stains  (e.  g.,  Ehrlich's  triacid  mixture  and  §  111). 
In  very  dilute  solutions  it  is  serviceable  in  staining  the  nuclei  of  fresh  tissue  and 
of  isolated  cells.  A  1  %  aqueous  solution  may  be  used  with  hematoxylin  and  picro- 
fuchsin  in  differentiating  the  structure  of  the  hair  follicle.  (Gage). 

§  110.  Safranin.  Formula  (Babe's):  Concentrated  aqueous  solution  of 
safranin,  1  part;  concentrated  alcoholic  solution  of  safranin,  1  part. 

Stain  sections  1  to  4  hours,  or  over  night;  wash  away  excess  of  stain  with 
95%  alcohol,  differentiate  with  acid  alcohol  (95%  alcohol,  100  c.  c.,  hydrochloric 
acid,  1/10  c.  c.)  for  a  few  seconds,  rinse  with  95%  alcohol  and  clear  in  carbol- 
xylene  or  bergamot  oil.  If  a  pure  nuclear  stain  is  not  required,  the  differentiation 
may  be  omitted.  This  gives  a  good  stain  with  tissue  fixed  in  Hermann's  or 
Flemming's  fluid.  It  is  a  brilliant,  transparent  red. 

Other  formulas  may  be  employed  (concentrated  alcoholic  solution,  alcoholic 
solution  diluted  with  anilin  water,  equal  parts  concentrated  solutions  in  alcohol 
and  anilin  water,  etc.).  Differentiation  may  be  accomplished  with  the  use  of 
iodin-potassium  iodid  solution,  or  by  counterstaining  with  an  alcoholic  solution 
of  light  green  or  acid  violet  (§  §  121,  122). 

§  111.  Ehrich-Biondi-Heidenhain  Mixture.  Formula:  Saturated  aqueous 
solutions  of  Orange  G.  Rubin  S.  (Fuchsin  acid)  and  Methyl  green,  100  c.  c.,  20  c.  c. 
and  50  c.  c.  respectively.  In  preparing  the  mixture,  only  fully  saturated  solutions 
should  be  taken  which  should  be  mixed  in  the  order  given  slowly  with  constant 
agitation.  Only  tissue  that  has  been  fixed  in  sublimate  solutions  (§11)  should 
be  used,  the  sections  should  be  thin  and  slightly  acid.  This  may  be  secured  by 


43 

treating  with  l/10th  per  cent,  acetic  acid  before  staining.  The  stain  is  useful  for 
some  cytological  work  (nuclear  degenerations,  cytoplasmic  transformations,  etc.). 
In  staining  use  the  stock  solution  diluted  with  distilled  water  1 :60  and  rendered 
slightly  acid  by  the  addition  of  0.2%  acetic  acid,  drop  by  drop,  until  the  red  color 
tone,  due  to  the  fuchsin  acid,  becomes  slightly  accentuated.  The  success  of  the 
stain  depends  upon  having  the  staining  reaction  right;  if  too  acid,  the  fuchsin 
acid  predominates,  otherwise  the  green  and  orange  prevail.  Stain  6  hours  or 
more;  when  sufficient,  dehydrate  rapidly  with  absolute  alcohol,  clear  with  xylene, 
mount  in  xylene  balsam. 

§  112.  Flemming's  Triple  Stain.  Stain  sections  in  an  alcoholic  solution  of 
safranin  diluted  with  an  equal  volume  of  anilin  water,  for  a  day  or  longer.  Differ- 
entiate in  absolute  (or  95%)  alcohol  with  l/10th  %  Hcl  until  hardly  any  more 
color  comes  away.  Stain  for  1  to  3  hours  in  a  1%  aqueous  solution  of  gentian 
violet.  Rinse  in  distilled  water  and  treat  with  a  strong  (2%)  solution  of  Orange  G. 
in  distilled  water,  and  while  clouds  of  violet  are  still  being  given  off,  bring  the 
sections  into  absolute  alcohol  in  which  the  differentiation  is  begun.  Transfer  the 
sections  to  clove  oil  or  bergamot  oil  which  completes  the  differentiation  and  clears. 
Mount  in  balsam  before  the  last  pale  clouds  of  color  have  ceased  to  come  away. 

This  stain  is  only  recommended  after  such  fixers  as  Flemming's  fluid  or  Her- 
mann's fluid.  The  stain  is  somewhat  fickle,  giving  the  best  results  only  after  some 
practice.  It  is  useful  in  some  cytological  work. 

Several  modifications  have  been  proposed.  A  short  method  is  frequently 
employed  as  follows:  (1)  stain  for  a  second  or  two  in  a  mixture  of  equal  parts  of 
saturated  aqueous  and  saturated  alcoholic  solutions  of  safranin;  (2)  rinse  in 
water;  (3)  stain  2  to  10  minutes  in  1%  gentian  violet;  (4)  rinse  in  water;  .and  (5) 
stain  for  10  seconds  or  longer  in  a  2%  solution  of  orange  G..  Dehydrate  rapidly 
with  absolute  alcohol;  clear  and  differentiate  with  clove  oil,  controlling  the 
differentiation  under  the  microscope.  Remove  the  clove  oil  with  xylene  or  toluene 
and  mount  in  balsam. 

§  113.  Congo  Red.  A  well  known  indicator,  red  in  neutral  or  alkaline 
solutions,  turning  blue  in  the  presence  of  free  mineral  and  many  organic  acids, 
not  affected  by  acetic,  lactic  or  carbonic  acid  in  the  presence  of  ammonia.  It  is 
useful  as  a  plasma  stain  after  hematoxylin,  gentian  violet,  etc. 

It  may  be  employed  in  aqueous  (^2  %)  or  alcoholic  (2%)  solution.  With 
subsequent  differentiation  in  acid  alcohol  (§  110)  it  is  a  useful  stain  with  gastric 
glands.  Occasionally  useful  as  an  indicator  with  living  organisms  or  tissue. 

§  114.  Eosin.  Formulas:  (a)  %%  aqueous  solution;  (b)  2-%  aqueous 
solution;  (c)  1/10%  solution  in  water  or  95%  alcohol.  Formula  (a)  is  preferable 
for  most  work;  (b)  affords  a  stronger  and  (c)  a  weaker  stain.  This  may  be  used 
as  a  counter-stain  with  hematoxylin  to  differentiate  nucleus  from  cell-body. 
Stain  sections  after  hematoxylin  for  10  to  30  seconds,  wash  away  the  excess  of 
stain  with  distilled  water  or  67%  alcohol.  Since  alcohol  tends  to  wash  out  the 
eosin,  unless  the  color  is  too  strong  it  is  advisable  to  hasten  the  process  of  washing 
out  and  dehydration. 

§  115.  Erythrosin.  Formulas:  (a)  l/z  to  1%  solution  in  67%  alcohol, 
(b)  ^4  to  1%  aqueous  solution.  This  is  a  general  stain  similar  to  eosin  in  its 


44 

staining  properties,  but  gives  a  redder  color.     Formulas  (a)  and  (b)  may  be  used 
with  sections  and  in  the  same  way  as  eosin. 

§  116.  Picric  Acid.  A  counter-stain  useful  after  carmine  or  hematoxylin. 
Use  ay£tol%  solution  in  67%  or  95%  alcohol,  or  simply  add  it  to  the  alcohol  used 
in  dehydration.  It  washes  out  the  hematoxylin  and  is  useful  as  a  differentiator 
of  this  stain,  with  which  it  is  well  to  overstain  somewhat  if  it  is  desired  to  counter- 
stain  with  picric  acid. 

§  117.  Orange  G.  An  excellent  acid  stain  that  may  be  employed  as  counter- 
stain  and  differentiator  after  hematoxylin,  gentian  violet,  etc. 

Employ  (a)  2  to  4%  aqueous  solution  which  may  with  advantage  be  slightly 
acid  (Hcl.) ;  in  the  last  event,  sections  should  be  rinsed  well  before  the  mounting. 
It  may  with  good  effect  be  combined  with  eosin,  erythrosin  or  fuchsin  acid,  (b) 
Frequently  a  concentrated  solution  in  95%  alcohol  is  to  be  preferred  to  the 
aqueous  solution. 

§  118.  Fuchsin  Acid  (Rubin  S.,  Magenta  S.).  Like  Congo  red,  an  indi- 
cator, red  in  acid  or  neutral  solutions,  bleached  by  alkali.  It  is  a  valuable 
plasma  stain,  but  requires  care  in  its  use  because  of  its  sensitiveness  to  alkali. 
Tap  water  should  therefore  be  avoided.  The  staining  solution  and  the  mounting 
medium  should  preferably  be  slightly  acid, — at  least  not  alkaline. 

Employ  a  2  to  4%  solution;    stain  1  minute  to  24  hours. 

§  119.  Picro-fuchsin.  Formulas:  (a.)  General  stain, — 1%  aqueous  solution 
of  fuchsin  acid,  10  c.  c.;  saturated  aqueous  solution  of  picric  acid,  75  c.  c.;  dis- 
tilled water,  25  c.  c.  (b)  For  nervous  tissue, — 1%  aqueous  solution  of  fuchsin 
acid,  15  c.  c. ;  saturated  aqueous  solution  of  picric  acid,  50  c.  c. ;  distilled  water, 
50  c.  c.  This  is  a  valuable  counter-stain  to  hematoxylin,  especially  serviceable 
in  the  differentiation  of  white  connective  tissue  fibers.  The  nuclei  are  a  purplish 
brown  (hematoxylin  stain),  the  connective  tissue  red,  cell  bodies  and  muscle 
yellow-orange.  In  special  cases  the  relative  amount  of  fuchsin  acid  may  be 
decreased  or  increased,  thus  giving  a  preponderance  to  the  yellow  or  red  in  the 
general  stain. 

Stain  well  with  hematoxylin,  rinse  in  water,  and  stain  with  the  picrofuchsin 
15  to  30  seconds;  wash  away  the  excess  of  stain  with  distilled  water  or  67% 
alcohol.  Picro-fuchsin  will  gradually  wash  out  the  hematoxylin,  therefore  stain 
strongly  with  hematoxylin  and  regulate  carefully  the  time  of  staining  with  picro- 
fuchsin. Picro-fuchsin  is  quite  sensitive  to  alkalies,  so  that  tap-water  (unless 
slightly  acidulated)  should  not  be  used  for  washing  out  and  the  mounting  medium 
should  be  slightly  acid  or  neutral,  not  alkaline. 

§  120.  Mallory's  Anilin  Blue  Connective  Tissue  Stain.  Fix  tissue  in  Zenker's 
fluid  (preferred)  or  in  a  mercuric  chlorid  fixer;  imbed  preferably  in  paraffin. 

Sections  are  to  be  stained  (a)  for  5  minutes  or  longer  in  a  1/5%  aqueous  solu- 
tion of  acid  fuchsin,  and  (b)  without  washing,  stained  about  four  times  as  long  in 
the  following:  Griibler's  water  soluble  anilin  blue,  0.5  gram;  orange  G.,  2.0 
grams;  1%  aqueous  solution  of  phosphomolybdic  acid.  100  c.  c.  Wash,  differ- 
entiate and  dehydrate  with  95%  and  absolute  alcohol.  Clear  in  xylene  and 
mount  in  balsam. 


45 

A  stain  of  quite  general  usefulness.  The  collaginous  connective  tissue  fibers,, 
reticular  tissue,  cartilage,  osseous  tissue,  mucus,  etc.,  are  stained  blue;  nuclei,, 
cytoplasm,  muscle,  red;  red  blood  corpuscles,  orange. 

§  121.  Light  Green.  An  acid  stain  sometimes  useful  as  a  counterstain  with 
safranin.  A  0.2%  alcoholic  solution  may  be  used  in  the  differentiation  of  the 
latter  stain. 

§  122.     Acid  Violet  may  be  used  in  similar  cases  and  in  the  same  manner^ 

§  123.  Bleu  de  Lyon  (Lyons'  Blue).  A  plasma  stain  frequently  useful  as  a 
counter  stain  after  safranin  or  carmine  (in  toto).  Employ  an  alcoholic  (95%) 
solution  of  about  half  saturation  and  stain  sections  for  10  to  15  minutes  or  longer. 
Rinse  with  95%  alcohol  and  dehydrate  more  or  less  rapidly  depending  on  the 
differentiation  desired.  • 

§  124.  Iodine.  In  addition  to  a  certain  value  as  a  fixer  and  to  facilitate  the 
removal  of  mercuric  chlorid  from  tissue,  usefulness  as  a  differentiator  after  basic 
aniline  dyes,  iodine  has  a  place  among  the  stains  for  the  differential  coloring  of 
starch,  glycogen,  amyloid,  cellulose,  etc.  The  following  solutions  may  be  men- 
tioned: (a)  saturated  aqueous  solution;  (b)  Iodine-potassium  iodid  solutions. 
(Gram's,  Lugols), — iodine  1  gram,  potassium  iodid  2  grams,  distilled  water  300  c.  c 
Lugol's  solution  is  six  times  as  strong,  (c)  10%  alcoholic  solution,  (tincture); 
(d)  for  a  formula  useful  for  staining  glycogen,  amyloid,  etc.,  see  §  229. 

§  125.  Resorcinfuchsin  (Weigert).  Prepare  a  concentrated  solution  of 
the  dry  powder  in  a  1%  solution  of  hydrochloric  acid  in  95%  alcohol.  Designed 
for  staining  differentially  elastic  connective  tissue  fibers. 

Stain  sections  15  to  30  minutes,  wash  and  dehydrate  in  95%  and  absolute 
alcohol,  clear  in  xylene,  mount  in  balsam. 

The  stain  may  be  directly  prepared  according  to  a  modified  formula  of  Weigert,, 
Formula:  Fuchsin  (basic),  2  grams;  resorcin,  4  grams;  water,  200  c.  c.  Boil 
several  minutes  (10  or  more);  add  25  c.  c.  30%  solution  ferric  chlorid  and  boil 
5  or  10  minutes  longer.  If  the  stain  is  not  all  precipitated,  more  of  the  ferric 
chlorid  solution  may  be  added.  Permit  the  liquid  to  cool.  Let  the  precipitate 
settle  and  decant  fluid,  or  filter.  Dissolve  precipitate  in  200  c.  c.  95%  alcohol, 
employing  heat  if  desired  (boiling  on  a  water  bath).  Filter.  When  the  filtrate 
is  cool,  add  4  c.  c.  strong  hydrochloric  acid. 

The  stain  may  be  followed  by  picro-fuchsin  or  other  similar  reagent  for  stain- 
ing the  white  connective  tissue  fibers. 

§  126.  Orcein  (Taenzer-Unna).  Another  standard  method  of  staining 
the  elastic  connective  tissue  fibers  (elastin).  Formula:  Orcein  D.,  1  gram;: 
95%  alcohol,  100  c.  c.;  strong  hydrochloric  acid,  1  c.  c. 

Stain  sections  ]/2  to  1  hour,  wash  and  differentiate  in  95%  alcohol  and  acid 
alcohol  (§  82).  Elastin  stained  dark  brown.  Other  differential  stains  may  be 
used  on  the  same  preparation. 

§127.  Verhoeff's  Elastin  stain.  [44]  Formula:  Hematoxylin,  0.15  grams;. 
Absolute  alcohol,  25  c.  c.;  dissolve  by  heat  and  add  1  drop  of  5%  ammonia  solu- 
tion. Permit  it  to  stand  for  at  least  5  minutes  and  add  Lugol's  solution  (§  124),. 
22  c.  c. ;  filter  and  permit  it  to  stand  for  24  hours  in  a  corked  bottle.  The  solution 
remains  good  for  about  3  months.  For  use,  add  a  7%  ale.  sol.  ferric  chlorid,  1 
drop  per  c.  c. 


46 

Stain  sections  from  alcohol,  12  to  24  hours;  wash  in  water  1  to  5  minutes; 
differentiate  in  a  1%  ferric  chlorid  solution  (ale.  stock  solution,  1  part,  water  6 
parts).  Counter  stain  with  picric  acid  (§116).  A  sharp  stain,  though  possibly 
not  in  all  cases  completely  differential. 

128.  Jenner's  Stain  [27].  This  is  an  eosinate  of  methylene  blue.  Formula: 
Mix  equal  parts  of  1.25%  eosin  (water  soluble)  and  1%  methylene  blue.  Permit 
the  mixture  to  stand  for  24  hours,  then  filter,  wash  the  precipitate  on  the  filter 
with  distilled  water,  dry  it  and  dissolve  it  in  methyl  alcohol  (0.5  grm.  precipitate 
in  100  c.  c.  alcohol). 

This  is  primarily  a  stain  for  blood  films.  Sections  may  be  stained  in  one  of  two 
ways:  (a)  Stain  1  to  5  minutes,  dilute  the  stain  with  an  equal  volume  of  water 
and  continue  the  staining  for  5  to  10  minutes.  Rinse  with  distilled  water,  differ- 
entiate and  dehydrate  rapidly  with  95%  and  absolute  alcohol.  Clear  in  xylene. 
Mount  in  balsam. 

(b)  Dilute  the  stain  with  1  to  4  volumes  of  distilled  water  and  stain  1  to  24 
hours.  Further  treatment  as  above.  To  insure  permanency,  it  is  well  to  thor- 
oughly remove  the  alcohol  with  xylene  in  clearing,  by  2  or  3  treatments. 

§  129.  Wright's  stain.  Contains  several  staining  principles,  notably  eosin- 
ates  of  methylene  blue,  methylene  azure,  in  solution  in  methyl  alcohol,  and  is 
made  according  to  a  complicated  method,  for  which  see  original  article  [46]. 

Primarily  for  blood  films  (§  214).  Dissolve  0.2  grams  of  the  dry  stain  in  100 
c.  c.  of  methyl  alcohol.  In  staining  sections  follow  the  procedure  given  in  §128. 

§  130.  Nochts-Hastings  Stain  [22].  Similar  in  its  composition  to  Wright's 
stain  and  used  in  a  similar  manner.  In  staining  sections,  follow  the  procedure  given 
in  §  128.  Dissolve  0.3  grams  of  the  dry  stain  in  100  c.  c.  of  methyl  alcohol.  An 
excellent  blood  stain  (§  214). 

§  131.  Neutral  Gentian  Violet  (Reinke,  Bensley).  Formula:  Saturated 
aqueous  solution  of  gentian  violet  and  saturated  solution  of  orange  G.  are  mixed 
in  equal  proportions  and  permitted  to  stand  24  hours.  The  precipitate  that  forms 
is  removed  by  filtration,  washed  with  distilled  water,  dried,  and  a  concentrated 
solution  in  methyl  or  ethyl  alcohol  is  made.  When  ready  to  use  it,  dilute  1  to  4 
times  with  distilled  water.  Stain  12  to  24  hours,  drain  away  the  stain,  dehydrate 
rapidly  with  95%  and  absolute  alcohol,  continuing  the  differentiation  if  necessary 
with  oil  of  cloves,  to  the  right  degree.  Remove  the  oil  of  cloves  with  xylene 
(thoroughly)  and  mount  in  balsam. 


PREPARATION    FOR    STAINING. 

§  132.  After  the  sections  are  cut  and  before  the  process  of 
staining  can  begin,  certain  steps  are  necessary,  such  as  the  removal 
of  the  paraffin  in  the  case  of  paraffin  sections.  Furthermore,  it  is 
usually  advisable  to  fasten  the  delicate  sections  to  the  glass  slides  on 
which  they  are  to  be  finally  mounted  before  beginning  the  series  of 
manipulations  that  are  necessary  in  Staining  and  Mounting.  To 
consider  the  latter  first: 


47 

§  133.  Handling  the  Sections.  Sections  may  be  carried  on 
through  the  staining  and  mounting  processes  either  (a)  not  fastened 
to  the  slide, — as  free  or  loose  sections,  or,  (b)  fastened  to  the  slide, 
which  is  of  the  greatest  advantage  and  practically  necessary  in  the 
case  of  serial  sections.  The  methods  of  fastening  the  sections  to  the 
slide  are  different  for  celloidin  and  paraffin  sections. 

§  134.  Free  sections.  It  is  seldom  necessary  or  advantageous 
to  carry  paraffin  sections  through  the  processes  of  staining  and 
mounting  not  attached  to  slides  because- of  their  delicacy  and  the 
readiness  with  which  they  tear  or  fray.  Celloidin  sections  may  be 
more  conveniently  carried  on  in  this  way.  Loose  sections  may  be 
carried  on  in  watchglasses  or  larger  glass  vessels  if  there  are  many 
of  them,  the  sections  either  being  transferred  from  vessel  to  vessel 
by  means  of  forceps  or  a  section  lifter,  or  the  fluid  decanted,  care 
being  taken  not  to  pour  off  the  sections,  and  the  succeeding  medium 
added.  Single  sections  may  be  best  carried  on  upon  the  slide  which 
must  be  kept  horizontal.  When  the  fluid  is  to  be  changed,  place  a 
brush  or  needle  gently  on  one  corner  of  the  specimen  and  pour  off 
the  liquid,  if  necessary  first  absorbing  most  of  it  by  means  of  a 
pipette;  in  this  way  the  section  may  be  retained  on  the  slide.  If 
many  sections,  not  in  series,  are  to  be  treated  in  the  same  manner, 
they  may  be  placed  in  a  perforated  container, — box  or  basket, — 
and  handled  as  a  unit  by  transferring  the  container  from  fluid  to 
fluid  (§  §  144—).  When  the  final  step  is  reached  (§  159)  the  sec- 
tions may  be  transferred  to  slides  and  mounted. 

§  135.  Fastening  sections  to  the  slide.  1.  Celloidin  sections. 
Sections  cut  by  either  the  alcohol  or  the  clarifier  methods  may  be 
conveniently  fastened  to  the  slide  in  the  following  manner: 

If  the  sections  are  transferred  to  the  slide  from  clarifier  or  clearer, 
absorb  the  fluid  thoroughly  by  placing  over  the  section  some  absorbent 
paper  and  pressing  it  down  gently  and  firmly,  repeating  the  operation 
several  times  with  fresh  paper.  After  the  oil  is  well  absorbed,  with 
a  pipette  drop  upon  the  section  enough  ether-alcohol  to  thoroughly 
wet  it.  This  softens  or  dissolves  the  celloidin  and  on  its  evaporation 
the  section  sticks  to  the  slide.  Allow  the  ether-alcohol  to  evaporate 
until  the  celloidin  has  again  set  and  the  surface  of  the  section  looks 
dull  or  glazed  and  then  place  it  in  a  jar  of  95%  alcohol.  Do  not  let 
the  sections  dry. 

If  the  sections  are  in  series,  it  is  better  to  put  the  ether-alcohol 
on  one  end  of  the  slide  and  let  it  run  quickly  over  the  sections  and 


48 

drain  from  the  other  end  of  the  slide,  repeating  the  operation  two  or 
three  times  if  necessary.  If  the  sections  are  well  pressed  down  and 
the  clarifier  thoroughly  absorbed  the  sections  will  stick  to  the  slide 
under  most  rough  manipulation.  If,  however,  it  is  found  that  the 
sections  tend  to  float  off  of  the  slide  in  the  process  of  staining,  their 
adhesion  may  be  insured  by  using  albuminized  slides,  or  removing 
the  slides  from  the  alcohol  (§  144)  and  again  treating  with  ether- 
alcohol. 

Sections  cut  in  alcohol  may  require  more  ether-alcohol. 

§  136.  Celloidin  Sections  with  Removal  of  the  Celloidin.  In 
the  above  method,  the  celloidin  is  used  in  fastening  the  section  to 
the  slide.  As  celloidin  stains  heavily  with  most  basic  stains,  it  is 
sometimes  desirable  to  have  it  removed  as  is  paraffin.  In  most  such 
cases,  the  difficulty  may  be  avoided  by  employing  paraffin  as  the 
imbedding  method;  where  this  is  not  possible,  imbed  in  celloidin  as 
usual  but  harden  and  cut  in  alcohol.  Use  67%  alcohol  on  the 
microtome  knife,  handle  the  sections  with  tissue  paper  as  usual 
(§  70),  but  before  transferring  them  to  slide  previously  prepared 
with  albumen  fixative  (§  138),  let  as  much  of  the  alcohol  evaporate 
as  is  possible  without  the  sections  drying.  They  should  also  be  free 
from  wrinkles.  Press  the  sections  down  well,  remove  the  tissue 
paper  and  place  the  slide  in  95%  alcohol  which  will  harden  the 
albumen  fixative  cement.  The  celloidin  may  be  removed  now  or  just 
before  clearing  by  means  of  ether-alcohol,  absolute  alcohol,  or  clove 
oil. 

§  137.  Free-hand  Sections  and  such  made  with  the  freezing 
microtome  may  be  fastened  to  the  slide  if  desired  by  albumin  fixa- 
tive or  M%  celloidin  or  both.  If  the  first  is  used,  the  sections  should 
be  transferred  to  the  albuminized  slide  from  water  which  should  be 
largely  absorbed  and  the  sections  pressed  down  by  tissue  paper.  If 
the  latter,  press  sections  to  a  clean  slide  with  paper,  pour  on  95% 
alcohol,  dip  cautiously  into  %%  celloidin,  drain  and  place  slide  in 
95%  alcohol. 

§  138.  2.  Paraffin  Sections  are  most  conveniently  fastened 
to  the  slide  by  albumen  fixative  and  spreading  with  water,  as  fol- 
lows:— Place  upon  the  slide  a  small  drop  of  Mayer's  albumin  fixa- 
tive* and  with  a  (clean)  finger  tip  spread  it  into  a  thin  even  film. 
Place  upon  the  albuminized  surface  enough  distilled  water  to  float 


M*ayer's  Albumen  fixattie.     White  of  egg,  50  c.  c. ;  glycerin,  50  c.  c. ;  salicylate 
of  soda,  1  gram.     Shake  them  thoroughly  and  filter. 


49 

the  sections  freely.  Cautiously  and  slowly  warm  the  slide  over  a 
small  flame,  as  that  of  an  alcohol  lamp,  or  upon  a  metal  warming  table 
until  the  sections  begin  to  spread  and  straighten  out.  When  the 
wrinkles  have  entirely  disappeared,  allow  the  water  to  cool  and  then 
drain  it  off,  retaining  the  sections  in  position.  The  slide  should  now 
stand  2  to  3  hours  or  better  over  night  when  the  water  beneath  the 
sections  having  evaporated »  they  will  have  been  brought  close  to  the 
albumen  fixative.  They  are  now  ready  for  the  remaining  steps. 
Melt  the  paraffin  by  warming  the  slide  over  a  low  flame  and  place  it 
in  xylene  (§  §  143—). 

§  139.  Albumin  fixative  and  Heat.  If  the  sections  are  free 
from  wrinkles  or  with  few  wrinkles  that  can  be  easily  "ironed  out," 
place  the  sections  in  position  upon  a  slide  prepared  with  albumin 
fixative  as  above,  and  with  a  clean  finger  press  the  section  into  the 
albumin  fixative  beginning  at  one  edge  of  the  section  and  by  a  rolling 
motion  of  the  finger,  ironing  out  any  wrinkles  that  there  may  be. 
It  is  well  to  look  upon  the  reverse  side  of  the  slide  to  see  if  the  section 
really  adheres  to  the  albumen  fixative,  as  in  some  cases  it  does  not. 
Heat  the  slide  gently  and  slowly  over  a  small  flame  until  the  paraffin 
melts  and  begins  to  run  away  from  the  specimen.  Keep  the  paraffin 
just  melted  for  a  minute  or  so,  and  then  transfer  to  the  xylene. 
Should  the  paraffin  section  not  adhere  to  the  albumin  fixative  when 
well  pressed  down,  it  can  in  many  cases  be  made  to  do  so  by  briskly 
rubbing  the  reverse  side  of  the  slide  with  a  woolen  or  silk  cloth. 

§  140.  J4%  Celloidin  (collodion).  The  adhesion  of  sections 
that  are  particularly  valuable  or  relatively  thick  may  be  ensured  by 
treating  them  with  %%  celloidin,  as  follows :  Fasten  the  sections  to 
the  slide  by  either  of  the  above  methods,  remove  the  paraffin  by 
xylene  (§  143),  and  then  after  draining  off  the  xylene  from  the  slide, 
10  to  13  seconds,  it  is  put  into  a  bottle  containing  J4%  celloidin.  In 
a  minute  or  more  the  celloidin  displaces  the  xylene  and  penetrates 
the  sections.  The  slide  is  removed,  allowed  to  drain  for  half  a  minute 
and  then  put  into  a  jar  of  67%  alcohol  which  sets  the  celloidin  [17]. 
It  is  now  ready  for  the  staining  processes  (§  144 — ). 

PRELIMINARY    STEPS. 

§  141.  These  are  somewhat  different  for  paraffin  and  celloidin 
sections.  In  the  case  of  the  former,  it  is  necessary  to  remove  the 
paraffin  by  means  of  a  solvent  (e.  g.,  xylene),  remove  the  paraffin 


50 

solvent  by  alcohol,  and  usually  remove  the  alcohol  with  water.  In 
the  case  of  celloidin  sections  the  first  step  is  unnecessary.  If  the 
tissue  was  hardened  and  cut  in  alcohol,  the  second  step  may  likewise 
be  omitted. 

§  142.  In  toto  Staining.  If  the  staining  has  already  been 
done,  (§  79)  these  "preliminary  steps"  are  of  course  all  unnecessary, 
save  the  removal  of  the  paraffin  by  xylene. 

§  143.  Xylene.  Leave  paraffin  sections  in  xylene  until  the 
paraffin  is  entirely  dissolved  out,  requiring  usually  only  a  few  seconds. 
A  longer  stay  generally  does  no  harm. 

§  144.  Alcohol.  Transfer  paraffin  sections  from  xylene  to 
95%  alcohol  leaving  the  sections  in  the  alcohol  5  to  10  minutes;  or 
if  you  wish,  shorten  the  period  to  a  minute  or  so  by  waving  the  slide 
gently  to  and  fro  in  the  alcohol. 

Celloidin  sections  cut  by  the  clarification  method,  are  placed 
in  alcohol  to  remove  the  clarifier.  This  may  take  a  longer  time,  and 
if  there  are  many  slides  it  is  well  to  use  two  changes  of  alcohol.  A 
longer  stay  in  alcohol  does  no  harm. 

§  145.  Water.  Remove  the  95%  alcohol  with  water  if  the 
stain  is  an  aqueous  one. 

§  146.  Staining.  The  following  schema  shows  the  general 
steps  in  staining  and  mounting.  In  all  the  processes,  seemingly 
complicated,  if  it  is  remembered  that  the  succession  of  media  as  in 
histological  technique  generally  depends  upon  their  miscibility  or 
some  special  reaction,  and  the  reason  for  the  various  steps  is  recog- 
nized, much  of  the  difficulty  in  remembering  the  order  in  which 
they  come  will  be  avoided. 


51 


SCHEMA    FOR    THE    STAINING    OF    SECTIONS. 

Celloidin  Sections  Paraffin  Sections 


fasten  to  slide  not  fastened  to  slide 

I 

by 

(a)  absorbing  clarifier 

I 

and 

} 

(b)  flooding  with  a  few 
drops  of  ether-alcohol 

xvlene*- 


95  %    alcohol 


water 

J 

Aqueous  stain 
(e.  g.,  hematoxylin) 


water 


Aqueous 
Counter-stain 
(e.  g.,eosinor 
picro-fuchsin) 

1 

water,  or  67%  alcohol 


Alcoholic 

Counter-stain 

(e.g.,  orange  G.) 


alcohol 


Dehydrate 
(95%-99%  alcohol) 

1 
Clear 

1 
Mount  in  balsam 


fasten  to  slide 

by 

albumen  fixative, 

spreading  with 

water  and  drying 


Alcoholic  stain 
(e.  g.,  Hcl.  carmine) 


alcohol 


52 

MOUNTING. 

§  147.  Whether  stained  or  unstained,  prepared  for  micro- 
scopical examination  by  isolation  or  sectioning,  and  especially  if  it 
is  desired  to  keep  the  preparation,  it  is  necessary  to  mount  it  in 
some  way, — i.  e.,  so  arrange  it  upon  some  suitable  support  (glass 
slide)  and  in  some  suitable  mounting  medium  that  it  may  be  satis- 
factorily studied  with  the  microscope. 
Mounting  may  be 

I.     Temporary,  or 
II.     Permanent, — as 

A.  Dry7,  or  in  air, 

B.  In  a  medium  miscible  with  water,  or 

C.  In  a  resinous  medium,  in  which  case  it  is  necessary 
first  to  remove  all  water  by  either  (a)   drying — Desiccation,  or  (6) 
a  series  of  displacements,  i.  e.,  I.     Removing  the  water  with  strong 
alcohol — Dehydration;     %.     Removing   the   alcohol   with   clearer — 
Clearing:,    3.     Replacing  the  clearer  with  balsam  or  other  resinous 
mounting  medium. 

§  148.  Temporary  mounting.  Illustrations  may  be  found  in 
the  examination  of  blood  corpuscles  and  living  ciliated  cells  (§  35). 
Temporary  examination  of  tissues  is  quite  simple,  though  important, 
and  for  this  it  is  only  necessary  to  place  the  teased  tissue  or  section 
on  the  slide  in  a  drop  of  the  fluid  in  which  it  is  at  the  time,  normal 
salt  solution,  dissociator,  or  alcohol,  and  cover.  The  examination  of 
preparations  intended  for  permanent  mounts  during  the  staining 
or  before  mounting  will  often  serve  to  detect  faulty  treatment  at  a 
time  when  it  may  be  remedied  without  great  expenditure  of  time,  or 
to  discard  the  specimen  as  worthless. 

§  149.  Permanent  mounting.  These  usually  include  (a)  mount- 
ing dry  on  a  ring  or  in  a  cell,  (6)  in  glycerin  or  glycerin  jelly,  media 
miscible  with  water,  and  (c)  in  Canada  balsam  or  damar,  resinous 
media. 

§  150.  Mounting  dry.  The  preparation,  may  be  either  upon 
the  under  side  of  the  cover-glass  (best  if  possible)  or  rest  upon  the 
bottom  of  the  cell. 

In  the  first  case  a  shallow  cell  made  by  a  shellac  ring  will  be 
sufficient;  in  the  second,  a  shellac  ring  may  not  give  a  deep  enough 
>  cell  and  a  paper,  hard  rubber,  or  metal  ring  may  be  cemented  to  the 
slide. 


53 

(a)  When  the  preparation  is  on  the  cover.     Prepare    a    shellac 
cell  (§  156)  on  the  slide  of  a  size  slightly  smaller  than  the  cover  to 
be  used,  and  allow  it  to  dry  for  a  day  or  so.     Warm  the  cover  bear- 
ing the  preparation  to  remove  the  last  traces  of  moisture,  and  place 
it  film  side  down  upon  the  ring.     Warm  the  slide  until  the  edge  of 
the  cover  may  be  made  to  adhere  to  the  shellac  ring  and  press  the 
cover  down  until  it  adheres  all  the  way  round.     Seal  the  cover  with 
shellac  and  label  (§  §  163,  165). 

(b)  Mounting  in  a  paper  or  rubber  cell.     With  a   brush,   cover 
one  side  of  the  ring  with  a  layer  of  shellac  and  place  it  on  the  center 
of  the  slide,  shellac  side  down;    place  within  the  cell  the  prepara- 
tion, arranging  it  in  the  manner  desired,  and  place  upon  the  ring  a 
cover-glass  of  a  suitable  size,  and  seal  it  with  shellac;   label. 

§  151.  Mounting  in  glycerin  media,  (a)  Pure  glycerin;  (b) 
glycerin  and  acetic  acid,  1%;  (c)  glycerin  and  a  stain.  As  glycerin 
extracts  most  stains  (Ex.  Carmine)  it  is  sometimes  advisable  to 
have  a  small  amount  of  stain  dissolved  in  the  glycerin  used  for 
mounting.  Of  such  combined  mounting  and  staining  mixtures  may 
be  mentioned  (1)  Glycerin  and  Congo  red,  (2)  Glycerin  and  car- 
mine. Other  combinations  may  be  used. 

§  152.  Congo  Glycerin.  Formula:  J/2%  aqueous  solution, 
Congo  Red,  1  part;  glycerin,  1  part. 

§  153.  Carmine  Glycerin.  Formula: — Carmalum,  25  c.  c., 
Glycerin,  75  c.  c. 

§  154.  Methyl  green  and  Eosin  Glycerin.  Formula: — 1% 
aqueous  solution  methyl  green,  2  c.  c.;  >£%  aqueous  eosin,  1  c.  c., 
glycerin,  97  c.  c. 

Glycerin  and  glycerin-jelly  are  most  serviceable  in  mounting 
isolation  preparations.  For  both  of  these  mounting  media  the  ob- 
ject must  be  mounted  from  water  or  an  aqueous  solution. 

Arrange  the  section  or  teased  tissue  in  the  center  of  the  slide, 
drain  off  the  water  or  aqueous  solution  in  which  the  preparation  is 
and  add  a  small  drop  of  glycerin.  Take  a  clean  cover  in  the  forceps, 
breathe  on  the  under  side  and  carefully  lower  it  upon  the  object; 
gently  press  it  down.  It  is  best  to  use  only  a  small  drop  of  glycerin 
so  as  not  to  get  it  outside  the  cover,  as  it  is  hard  to  clean 
away  satisfactorily.  Clean  carefully  and  seal  with  shellac  in 
accordance  with  §  163. 


54 

§  155.  Mounting  in  glycerin-jelly.  The  preparation  should 
be  mounted  from  some  aqueous  solution.  Warm  the  slide  gently 
and  put  it  upon  the  centering  card;  in  the  center  of  the  slide  place 
a  drop  of  warmed  (melted)  glycerin- jelly.  Remove  the  object  from 
the  water  or  aqueous  solution  and  arrange  it  in  the  glycerin -jelly. 
Grasp  a  cover-glass  with  the  fine  forceps,  breathe  on  the  lower  side, 
gradually  lower  it  upon  the  object  and  gently  press  it  down.  Allow 
the  glycerin-jelly  to  set,  keeping  the  slide  horizontal  meanwhile. 
Scrape  away  the  superfluous  glycerin- jelly  around  the  cover-glass 
and  seal  with  shellac  (§  163). 

§  156.  Preparation  of  shellac  mounting  cells.  Place  the  slide 
upon  the  turn-table  and  center  it  (i.  e.,  get  the  center  of  the  slide 
over  the  center  of  the  turn-table).  Select  a  guide  ring  on  the  turn- 
table which  is  a  little  smaller  than  the  cover-glass  to  be  used;  take 
the  brush  from  the  shellac,  being  sure  that  there  is  not  enough 
cement  adhering  to  it  to  drop.  Whirl  the  turn-table  and  hold  the 
brush  lightly  on  the  slide  just  over  the  guide  ring  selected.  An 
even  ring  of  the  cement  should  result.  If  it  is  uneven,  the  cement 
is  too  thick  or  too  thin  or  too  much  was  on  the  brush.  After  a  ring 
is  thus  prepared,  remove  the  slide  and  allow  the  cement  to  dry 
spontaneously,  or  heat  the  slide  in  some  way.  Before  the  slide  is 
used  for  mounting,  the  cement  should  be  so  dry  when  it  is  cold 
that  it  does  not  dent  when  the  finger  nail  is  applied.  A  cell  of  con- 
siderable depth  may  be  made  with  shellac  by  adding  successive  lay- 
ers as  the  previous  one  dries. 

§  157.  Mounting  in  balsam:  by  desiccation.  Certain  prepara- 
tions may  be  mounted  in  balsam,  by  drying,  the  method  of  desiccation 
(§  147),  e.  g.,  cover-glass  preparations  of  bacteria,  stained  cover-glass 
preparations  of  blood,  etc.  For  this  is  it  only  necessary  that  the  prep- 
aration be  absolutely  dry,  a  small  drop  of  balsam  placed  upon  it  or 
upon  the  under  side  of  the  cover-glass,  which  is  carefully  placed 
over  the  specimen  and  pressed  down. 

Mounting  in  balsam:  by  displacement.  Mounting  in  balsam  by 
desiccation  is  serviceable  for  but  few  preparations  in  histology,  and 
in  most  cases  the  removal  of  the  water  by  a  series  of  displacements  is 
resorted  to  (§  147).  For  this  the  following  steps  are  necessary: 
Dehydration,  Clearing,  Mounting  in  balsam. 

§  158.  Dehydration.  The  sections  are  entirely  freed  from 
water  by  the  use  of  95%  or  absolute  alcohol.  The  slide  or  free  sec- 


55 

tion  may  either  be  placed  in  a  jar  of  alcohol  or  alcohol  from  a  pipette 
be  poured  over  it.  Treat  the  preparation  to  be  mounted  for  5  to  15 
minutes.  The  thicker  the  section  the  longer  the  time  required; 
celloidin  sections  require  a  longer  time  than  paraffin  sections.  In 
any  case,  be  sure  that  the  dehydration  is  complete,  giving  a  longer 
rather  than  a  shorter  time,  and  then  clear. 

§  159.  Clearing.  This  is  accomplished  by  putting  the  slide  in 
a  jar  of  clearer  or  dropping  the  clearer  upon  the  section  from  a 
pipette.  When  the  section  is  cleared  it  will  be  transparent.  Test 
it  by  holding  it  against  a  dark  background;  if  it  is  not  cleared  it 
will  be  cloudy,  white,  and  opaque.  Carbol-xylene  (melted  carbolic 
acid,  1  part;  xylene,  3  parts);  xylene;  or  certain  essential  oils  (ori- 
ganum, thyme,  cajuput,  bergamot)  are  used. 

§160.  Mounting  in  balsam.  Drain  off  the  clearer  and  allow 
the  section  to  stand  until  there  appears  the  first  sign  of  dullness 
from  evaporation  of  the  clearer  from  the  surface.  Then  place  a 
small  drop  of  balsam  upon  the  section  or  upon  the  cover-glass  \vhich 
is  then  inverted  over  the  specimen. 

Remember  that  in  mounting  in  this  way  you  must  always  "De- 
hydrate, Clear  and  Mount  in  Balsam,'''  and  that  the  three  steps  are 
inseparable. 

§  161.  Natural  balsam  is  acid  in  reaction  due  to  organic  acids  contained. 
As  these  bleach  basic  dyes,  notably  hematoxylin,  it  is  well  to  use  for  most  purposes 
balsam  that  has  been  dried  out  and  redissolved  in  a  known  solvent,  such  as 
xylene  or  benzene.  Neutral  balsam  solutions  are  to  be  preferred.  Alkaline 
balsam  is  sometimes  preferable  for  some  hematoxylin  stains;  acid  balsam  in 
certain  other  cases  (fuchsin  acid,  injection  masses,  §  §  118,  217 — ).  Furthermore, 
according  to  Mann  [36] ,  such  solvents  as  xylene  readily  oxidize  with  the  formation 
of  acid  products.  For  delicate  work,  therefore,  it  is  probably  advisable  to  use  as 
thick  balsam  as  is  convenient,  and  avoid  inclusion  of  air  bubbles  in  the  mounting. 
Benzene  balsam  from  this  standpoint  is  preferable  to  xylene  balsam. 

Certain  resinous  mounting  media, — Camsal  Balsam,  Euparal, — have  been 
recently  prepared  in  which  specimens  may  be  mounted  direct  from  95%  alcohol; 
clearing  is  then  unnecessary,  of  course. 

SEALING    THE    PREPARATIONS. 

§  162.  Sealing  glycerin  mounted  specimens.  Wipe  away  the 
superfluous  glycerin  as  carefully  as  possible  with  a  moist  cloth  or  a 
piece  of  lens  paper.  Place  four  minute  drops  of  cement  carefully  at 
the  edge  of  the  cover  at  the  four  quarters  and  allow  them  to  harden 
for  half  an  hour  or  more;  these  will  anchor  the  cover-glass  and  the 


56 

preparation  may  then  be  placed  upon  the  turn-table  and  a  ring  of 
shellac  cement  put  round  the  edge  while  revolving  the  turn-table. 

§  163.  Sealing  glycerin- jelly  mounts.  Allow  the  glycerin- 
jelly  to  harden  for  12  hours  or  longer.  With  a  knife  scrape  away 
the  superfluous  jelly  and  then  carefully  wipe  around  the  coyer-glass 
with  a  cloth  moistened  with  water.  Place  the  slide  on  a  turn-table, 
carefully  center  the  cover-glass,  and  with  a  brush  seal  the  edge  of 
the  cover  by  a  ring  of  shellac  while  revolving  the  turn-table.  A 
second  coating  may  be  given  subsequently  if  needed,  after  the  first 
coat  has  dried. 

§  164.  Sealing  balsam  mounts.  This  is  necessary  only  with 
special  preparations,  and  should  in  any  case  be  done  only  after  the 
preparations  have  dried  out  for  several  weeks.  With  a  knife  scrape 
off  all  superfluous  balsam  from  around  the  cover-glass  and  wipe  it 
carefully  with  a  cloth  moistened  with  alcohol  or  benzin  (or  xylene). 
Seal  as  with  glycerin- jelly  mounts. 


LABELING   MICROSCOPIC    SLIDES. 


§  165.  Every  permanent  microscopic  preparation  should  be 
carefully  and  neatly  labeled  in  ink,  the  label  being  placed  upon  the 
right  hand  end  of  the  slide.  The  label  should  furnish  at  least  the 
following  information: 


EXAMPLE. 


(1)  The  number  of  the  prepara- 
tion, the   thickness  of  the 
cover-glass  and  of  the  sec- 
tion. 

(2)  The  name,  kind,  and  source 
of  the  preparation. 

(3)  The  fixer  and  the  stain. 

(4)  The  date  of  the  specimen. 


No. 


Ileum  of  Cat. 
Transection. 

Z.*H.&E. 

November,  1898. 


C.  15 

S. 


In  the  case  of  specimens  with  which  it  is  advantageous  to  have 
more  information  at  hand  a  second  label  may  be  placed  upon  the 
other  end  of  the  slide,  and  it  may  bear  the  following  information: 


*It  is  convenient  to  adopt  a  standard  system  of  abbreviations,  thus: — Z.  = 
Zenker's  fluid;  He.  =  Kelly's  fluid;  M.  =  mercuric  chlorid,  etc. ;  H.  =  Hema- 
toxylin;  E.  =  eosin,  etc. 


57 

(1)  Mode  of  fixation  (detail) . 

(2)  Imbedding  method. 

(3)  Stains  employed  (detail). 

(4)  Mounting  medium  (generally  not  necessary). 

(5)  Special  purpose  of  the  preparation. 

A  catalog  giving  the  full  data  of  the  specimen, — age,  condition 
of  the  animal,  mode  of  preparation  in  detail,  special  points  illus- 
trated, etc.,  is  valuable  particularly  in  special  investigations  and 
with  standard  specimens. 

Paper  labels  are  very  convenient  but  possess  the  disadvantage  that  they  are 
very  apt  to  come  off.  In  this  laboratory  it  is  the  custom  to  label  permanent 
preparations  by  means  of  a  writing  diamond  with  the  record  number  at  least. 

§  166.  A  carbon  ink  label  written  upon  the  slide  itself  is  fairly  durable  and 
may  be  made  as  follows:  First  coat  the  end  of  the  slide  with  a  thin  solution  of 
balsam  (xylene  or  benzene),  and  permit  this  to  dry  thoroughly.  Upon  this  surface 
it  is  possible  to  write  neatly  with  carbon  ink  and  after  the  ink  is  dry  a  second  coat- 
ing of  the  thin  balsam  or  of  shellac  may  be  given.  This  method  is  recommended. 

SLIDES    AND    COVERS. 

The  slides  and  cover-glasses  used  in  histological  work  are  often 
slightly  greasy  and  should  be  cleaned  before  using.  After  they  are 
cleaned  they  should  be  handled  by  the  edges  only,  or  with  forceps. 

§  167.  Cleaning  Slides.  For  ordinary  work  it  is  enough  to 
wipe  the  slides  out  of  clean  water  to  which  about  5%  ammonia  has 
been  added.  A  clean  glass  towel  free  from  lint  should  be  used  and 
after  the  slides  are  cleaned  they  should  be  stored  in  covered  glass 
jars  away  from  the  dust. 

§  168.  Cleaning  Cover-glasses.  Place  them  in  95%  alcohol 
to  which  1%  of  hydrochloric  acid  has  been  added.  A  clean,  soft 
cloth  such  as  an  old  linen  handkerchief,  gauze,  etc.,  should  be  used 
for  wiping  them.  In  wiping  a  cover-glass  "grasp  it  by  the  edge  with 
the  left  thumb  and  index.  Cover  the  right  thumb  and  index  with 
the  cleaning  cloth;  grasp  the  cover  between  the  thumb  and  index 
and  rub  the  surfaces  keeping  the  thumb  and  index  well  opposed  on 
directly  opposite  faces  of  the  cover  so  that  no  strain  will  come  upon 
it,  otherwise  the  cover  is  liable  to  be  broken."  (Gage,  17). 

Cover-glasses  when  cleaned  should  be  stored  in  covered  glass 
boxes,  or  in  Petrie  dishes. 

§  169.  Cleaning  Mixture  for  Glass.  For  special  purposes,  such  as  when  the 
slide  or  cover-glass  is  to  be  used  in  the  preparation  of  blood  smears,  the  cleaning 


58 

mixture  whose  formula  is  given  below  may  be  used.  Place  the  cover-glasses  in 
this  mixture,  one  by  one,  and  permit  them  to  remain  over  night  or  longer.  Rinse 
them  thoroughly  in  running  water  until  all  color  of  the  dichromate  has  disappeared 
rinse  them  again  in  distilled  water  and  transfer  to  95%  alcohol,  out  of  which  they 
may  be  wiped. 

§  170.  Dichromate  Cleaning  Mixture.  Formula: — Potassium  dichromate, 
200  grams;  water,  800  c.  c.;  strong  sulphuric  acid,  1200  c.  c.  Dissolve  the 
dichromate  in  the  water  by  the  aid  of  heat,  and  to  the  solution  add  slowly  the 
sulphuric  acid.  The  two  fluids  should  be  mixed  in  a  lead-lined  kettle  [17]. 

§  171.  Used  slides  and  cover-glasses,  vials  and  other  glassware  that  have 
been  used  with  balsam,  cedarwood  oil,  or  other  oily  substance,  etc.,  may  be 
cleaned  by  boiling  them  with  a  solution  of  strong  soap,  such  as  "gold  dust,"  one 
or  more  changes.  Used  xylene  or  toluene  is  sometimes  useful.  Slides  and  covers 
may  require  a  second  cleaning  with  the  cleaning  mixture.  If  only  water,  glycerin 
or  glycerin- jelly  has  been  used  on  them,  they  may  be  cleaned  with  water,  prefer- 
ably warm  water,  and  then,  if  necessary,  wiped  out  of  50%  alcohol. 

§  172.  Measuring  the  thickness  of  the  cover-glasses.  With  the  cover- 
glass  measurer  determine  the  thickness  of  the  cover-glasses  and  sort  them  into 
three  groups:  (a)  those  with  a  thickness  of  .13-.17  mm.,  (b)  those  less  than 
.13  mm.,  and  (c]  those  thicker  than  .17  mm.  Groups  (a)  and  (b)  only  should  be 
used;  (c)  should  be  discarded  or  used  only  with  objects  for  low  magnification. 

It  is  advantageous  to  know  the  thickness  of  the  cover-glass  on  an  object 
for  the  following  reasons:  (a)  That  one  do  not  try  to  use  objectives  in  studying 
the  preparation  of  a  shorter  working  distance  than  the  thickness  of  the  cover- 
glass  [17] ;  (b)  In  using  adjustable  objectives  with  the  collar  graduated  for  different 
thicknesses  of  cover,  the  collar  might  be  set  at  a  favorable  point  without  loss  of 
time;  (c)  For  unadjustable  objectives  the  thickness  of  cover  may  be  selected 
corresponding  to  that  for  which  the  objective  was  corrected  [17].  Furthermore  if 
there  is  a  variation  from  the  standard  one  may  remedy  it  in  part  at  least  by 
lengthening  the  tube  if  the  cover  is  thinner  and  shortening  it  if  the  cover  is  thicker 
than  the  standard  [17]. 

THE   MICROTOME    KNIFE. 

Finally,  the  microtome  knife  or  section  razor  should  receive  a 
passing  word  as  upon  it  depends  far  more  than  may  be  at  first  sus- 
pected the  excellence  of  results.  Scrupulous  care  should  be  taken 
to  maintain  a  keen  edge,  smooth  and  free  from  nicks  or  corrosion. 
Never  touch  the  edge  with  anything  hard  or  metallic.  Keep  it  clean. 
Before  and  after  using  it  is  advisable  to  strop  it  upon  a  strop  backed 
with  wood,  and  occasionally  it  will  be  necessary  to  hone  it. 

§  173.  For  microtome  knives  two  grades  of  hones  are  service- 
able; the  yellow  Belgian  for  first  sharpening  and  either  a  blue-green 
water  hone  or  a  fine  Arkansas  oil  stone  for  finishing.  These  should  be 
kept  clean  and  free  from  dust.  If  a  section  knife  is  used  a  great 


59 

deal,  it  is  best  to  put  it  on  the  fine  hone  for  a  short  time  before  each 
day's  sectioning.  Unless  experienced  in  sharpening  section  knives, 
it  is  well  for  the  first  few  times  to  work  under  the  direction  of  one 
skilled  in  the  manipulation.  There  are,  however,  excellent  accounts 
to  be  found  in  works  on  technique  [e.  g.,  17,  21a]. 

Do  not  be  satisfied  with  any  but  a  smooth  edge,  keen  enough  to 
cut  without  "pulling,"  a  hair  held  in  the  fingers,  a  quarter  of  an 
inch  or  more  from  the  fingers.  When  once  a  good  edge  is  secured, 
take  pains  to  preserve  it. 

With  the  yellow  hone,  use  a  lather  of  olive  oil  soap;  the  blue- 
green  soap  stone  is  rubbed  up  with  water;  with  the  Arkansas  stone 
apply  a  good  thin  oil. 


SPECIAL  METHODS. 

THE    CELL. 

The  technique  of  the  Cell  is  almost  coextensive  with  that  of  his- 
tology as  a  whole,  at  least  as  far  as  concerns  the  application  of  the 
more  exact  and  delicate  methods.  It  is  necessary  therefore  to  give 
here  only  the  more  salient  points  and  accepted  methods. 

§  174.  General  Methods.  Hermann's  fluid,  Flemming's  fluid, 
Zenker's  fluid,  Mercuric  chlorid,  Carney's  fluids,  and  Picro-aceto- 
formol  are  standard  fixers,  although  special  problems  may  demand 
other  combinations.  Iron  Hematoxylin,  the  Ehrlich-Biondi-Heiden- 
hain  triple  mixture,  Safranin,  Gentian  Violet  and  Orange  G.  are  per- 
haps the  most  serviceable  stains.  Of  these  iron  hematoxylin  is 
particularly  and  universally  useful.  The  Ehrlich  triple  stain  is 
valuable  in  the  more  analytical  work  and  should  follow  Mercuric 
chlorid  (or  Zenker's  fluid,  Carnoy's  fluid)  fixation.  Safranin  and 
gentian  violet,  separately  as  red  or  blue  stains,  or  successively  fol- 
lowed by  orange  G  (Flemming's  triple  stain)  only  after  Hermann's 
fluid,  or  Flemming's  fluid,  or  similar  mixtures. 

§  175.  Chromatin.  While  regressive  stains  of  the  iron  hema- 
toxylin type  give  valuable  chromatin  stains,  they  are  not  as  a  rule 
analytical  or  selective.  In  accordance  with  the  recommendation  of 
Heidenhain  it  is  better  to  stain  progressively  with  dilute  solutions 
if  a  pure  chromatin  (basichromatin)  stain  is  desired. 


60 

Methyl  green  is  one  of  the  most  delicate  and  precise  of  chro- 
matin  stains.  It  may  be  used  alone  or  in  combination,  as  in  the 
Ehrlich-Biondi  mixture  (§111).  A  very  dilute  hematoxylin  (§  88, 
92)  is  excellent. 

§  176.  Nucleoli.  The  Ehrlich-Biondi  may  be  recommended 
to  bring  out  other  nuclear  structures,  such  as  nucleoli,  although 
other  combinations  of  basic  and  acid  stains  may  be  used.  Mont- 
gomery recommends  Ehrlich's  hematoxylin  followed  by  a  strong 
aqueous  eosin. 

§  177.  Cytoplasm.  Quite  different  pictures  are  obtained  by 
the  use  of  fixers  as  similar  in  composition  as  Flemming's  fluid  and 
Hermann's  fluid,  the  difference  seeming  to  be  due  to  a  varying 
preservation  of  cytoplasmic  "granules."  The  ground  work  of  the 
cell  body,  the  so-called  Spongioplasm,  may  be  preserved  by  such 
fixers  as  Zenker's  fluid,  Flemming's  fluid,  Hermann's  fluid,  Carnoy's 
fluid,  etc.,  and  iron  hematoxylin  and  the  Ehrlich-Biondi  mixture  give 
satisfactory  stains.  The  granules  that  may  be  present  are  of  different 
kinds  and  often  not  easily  interpreted;  they  include, — (a)  "re- 
serve" material  (yolk  granules,  fat  granules,  etc.),  (b)  basophile, 
acidophile,  neutrophile  granules,  (c)  granules  less  easily  preserved 
which  include  many  secretion  granules,  etc. 

(a)  Yolk  granules.     If  present  in  large  amount  a  special  fixer 
may  be  indicated  (§  §  20,  21,  22). 

Fat  granules',    see  §  §  224. 

(b)  Basophile    granulations     (Granoplasma,     Unna),     may    be 
demonstrated  by  simple  alcohol  fixation  and  subsequent  differential 
staining  with  basic  dyes.     Compare  §  195  and  the  special  technique 
of  blood  (§  21 1-). 

(c)  These  require  special  technique:     in  general,  (1)  the  employ- 
ment of  oxidizers  such  as  dichromates,  osmic  acid,  formalin,   (2) 
no  acid,  or  a  minimum  amount.     This  seems  to  indicate  the  presence 
of  reducing  substances  usually  lipoid  in  nature  whose  combinations  are 
soluble  in  (or  rendered  soluble  by)  acid.     Here  belong  the  Mitochon- 
dria of  Benda. 

§  178.     Mitochondria.     Benda's  Method. 

1.  Fix  in  Flemming's  fluid  with  the  acid  reduced  to  3  drops;  2. 
Rinse  in  water,  1  hr.;  3.  Place  in  equal  parts  pyroligneous  acid  and 
1%  aqueous  solution  of  chromic  acid,  24  hours;  4.  2%  potassium 


61 

dichromate,  24  hours;  5.  Running  water,  24  hours;  6.  Alcohols; 
paraffin  imbedding;  7.  Sections  5^;  4%  ferric  alum  for  24  hours; 
8.  Rinse  in  distilled  water  and — 9.  Place  in  a  mixture  of  saturated 
alcoholic  solution  of  sodium  sulphalizarinate  1  c.  c.,  distilled  water,  80 
to  100  c.  c.,  24  hours;  10.  Rinse  away  the  stain  with  water;  11. 
Stain  on  the  slide  with  a  freshly  prepared  mixture  of  saturated  alcoholic 
solution  of  crystal  violet  and  aniline  water,  equal  parts,  warming 
until  it  steams;  12.  Rinse  in  w^ater  and  differentiate  in  30%  acetic 
acid  (1  minute  or  less) ;  13.  Rinse  in  running  water  5  to  10  minutes 
to  remove  the  acid,  drying  with  blotting  paper;  14.  Dehydrate 
rapidly  in  absolute  alcohol,  (dip);  15.  Clear  in  bergamot  oil  and 
xylene;  16.  Mount  in  neutral  balsam. 

The  above  method  is  capricious.  The  following  method  is  recom- 
mended: (1)  fixing  12  to  24  hours  in  Zenker's  fluid  or  the  Copper 
dichromate  mixture  (§  16)  with  1/10%  acetic  acid  only;  (2)  mor- 
danting 3  to  4  days  in  2.5%  dichromate  (or  Muller's  fluid  or  Erlicki's 
fluid  respectively);  (3)  paraffin  imbedding;  (4)  using  iron  or  copper 
hematoxylin  as  the  stain  (§  §  94,  95).  Benda's  fluid  may  be  substi- 
tuted for  the  fixer,  and  a  number  of  other  stains  may  be  used;  con- 
sult [6,  30]. 

§  179.  Secretion  Granules  in  the  cytoplasm,  such  as  the  zymogen 
granules  in  the  stomach  and  pancreas,  granules  of  the  suprarenal 
medulla  and  Islands  of  Langerhans,  etc.,  may  be  preserved  and 
demonstrated  in  the  same  manner  and  probably  for  the  same  general 
reasons ;  thus : — 

1.  Trypsinogen  granules.     Fix   in   1%   osmic   acid   which    pre- 
serves and  browns  them;    or  as  under  2  below. 

2.  Pepsinogen  granules.     Fix  in  Kelly's  fluid  or  one    of  sim- 
ilar  composition.     Iron   hematoxylin   or   Weigert's   copper   hema- 
toxylin, or  neutral  stains  (§  §  107,  128,  131)  may  be  used. 

3.  Medulla  of  suprarenal.     Apply  similar  fixation  methods,  e.  g., 
Kelly's  fluid,  and  regressive  staining  with  basic  stains  (e.  g.,  iron  or 
copper  hematoxylin,  toluidin  blue,  etc.). 


CONNECTIVE    TISSUE 

§  180.  White  (Collagenous)  Fibers.  Fuchsin  acid  is  particularly 
valuable.  Three  methods  of  applying  it  for  the  differential  staining 
of  connective  tissue  follow : 


62 

(a)  Picro-fuchsin.     See  §  119.     This  may  be  used  with  or  with- 
out a  basic  counter-stain  which  should  precede  it.     If  a  counter- 
stain  is  used  remember  to  overstain  and  use  the  picro-fuchsin  to  dif- 
ferentiate it. 

(b)  Orange-fuchsin.     Formula:     Fuchsin  acid,  2  grams;   Orange 
G.,  1  gram;   Glycerin,  7  c.  c.;   distilled  water,  100  c.  c.     Fix  tissue  in 
Flemming's  fluid.     Stain  sections  30  seconds;    dehydrate,  clear,  and 
mount  in  balsam  (not  alkaline) .     Suitable  also  for  staining  the  reticu- 
lar  tissue  (lymphatic  tissue). 

Somewhat  more  delicate  than  the  picro-fuchsin. 

(c)  Mallory's  connective  tissue  stain.     See  §  120.     While  not  a 
differential  stain  for  collaginous  fibers,  it  is  nevertheless  a  valuable  one, 
and  frequently  to  be  preferred. 

§  181.  Elastic  Fibers  (Elastin).  Employ  either  the  Weigert 
ResorchvFuchsin  or  the  Orcein  methods  (§  §  125,  126),  or  the  Verhoeff 
[44]  method  (§  127). 

Both  the  white  and  elastic  fibers  may  be  stained  in  the  same 
preparation,  the  elastic  fibers  being  stained  first. 

In  a  mixture  composed  of  white  and  elastic  fibers,  picro-fuchsin 
(§  119)  will  stain  the  elastic  fibers  a  light  yellow,  the  white  fibers  being 
colored  red. 

In  studying  the  connective  tissues,  it  should  be  remembered 
that  acetic  and  the  mineral  acids  cause  swelling  or  solution  (gelatini- 
zation)  of  the  white  fibers,  depending  upon  their  strength.  While 
this  improves  the  cutting  quality  of  organs  rich  in  connective  tissue, 
it  also  causes  vagueness  in  outline  of  the  white  connective  tissue 
fibrils.  It  may  therefore  be  advisable  to  decrease  the  percentage  of 
acetic  acid  in  the  fixer  when  the  connective  tissue  is  under  investi- 
gation. 

§  182.  Reticular  Tissue.  Fuchsin  acid,  Mallory's  connective 
tissue  stain,  or  the  orange-fuchsin  acid  mixture  may  be  chosen  for 
staining  this  form  of  connective  tissue.  As  however  the  cellular  ele- 
ments usually  mask  the  fiber  relations,  if  a  view  of  the  latter  is  desired 
the  cells  must  be  removed, — by  a  mechanical  method,  such  as  cau- 
tiously brushing  the  section  with  a  camel's  hair  brush,  or  by  digestion. 

§  183.  Digestion  Method.  The  organ  to  be  examined  (e.  g., 
lymphatic  node,  spleen,  etc.),  is  preserved  in  67%  alcohol,  cut  into 
sections  2  mm.  or  more  in  thickness,  washed  thoroughly  in  water  to 
remove  the  alcohol,  and  digested  with  pancreatic  solution  in  1% 


63 

sodium  carbonate  solution  in  an  incubator  at  38  C.  until  examination 
under  the  microscope  shows  that  the  cells  have  become  disintegrated 
and  digested.  The  digestion  fluid  should  be  changed  every  day  or 
2  or  3  days.  A  varying  length  of  time  is  necessary, — sometimes  a 
month  or  more.  Paraffin  sections  may  also  be  submitted  to  digestion, 
with  or  without  the  removal  of  the  paraffin. 

The  following  method  is  recommended  for  the  further  preparation 
of  digested  tissue.  After  washing  thoroughly  in  running  water  to 
remove  the  digestion  fluid,  the  tissue  is  carefully  imbedded  in  celloidin 
(§  61 — )  and  sectioned,  the  sections  being  20  to  30^  thick.  These 
sections  are  not  fastened  to  the  slide,  but  after  having  been  brought 
into  95%  alcohol  are  placed  in  a  concentrated  solution  of  acid  fuchsin 
in  95%  alcohol  to  which  a  drop  or  two  (per  50  c.  c.)  of  glacial  acetic  acid 
has  been  added.  After  several  minutes,  the  sections  are  rinsed  in 
95%  alcohol  to  remove  the  excess  stain,  cleared  in  carbol-xylene  and 
mounted  in  balsam.  By  this  method  the  delicate  morphology  is 
preserved  and  a  sharp  stain  is  secured. 

The  method  of  artificial  digestion  with  trypsin  or  pepsin  has 
other  applications  in  the  histological  analysis  and  it  possesses  a  dis- 
tinct value. 


CALCIFIED     STRUCTURES — BONE    AND    TEETH. 

(A) .     Decalcification . 

§  184.  For  the  purpose  of  investigating  the  soft  structures  of 
tissues  containing  lime  salts,  such  as  bone,  teeth,  and  calcified  carti- 
lage, it  is  necessary  to  remove  the  lime  salts  before  sections  can  be 
prepared  in  the  usual  way  by  a  process  known  as  decalcification. 
Solutions  of  a  large  number  of  acids,  combined  or  uncombined  with 
other  substances,  may  be  used  as  decalcifiers.  Very  satisfactory 
are:  (1)  Hydrochloric  acid,  1  c.  c.,  67%  alcohol,  100  c.  c. 

(2)  nitric  acid,  3  c.  c.;   70%  (67%)  alcohol,  97  c.  c.,  and 

(3)  nitric  acid,  5  c.  c.;    saturated  aqueous  solution  of  (potash) 
alum,  50  c.  c.;    water,  50  c.  c. 

In  the  first  and  second  formulas  the  alcohol,  in  the  third  the 
alum  acts  as  a  restrainer  of  the  acid.  The  first  or  second  of  these 
formulas  is,  perhaps,  better  for  bone;  the  second  has  a  more  rapid 
action  and  is  possibly  a  better  decalcifier  for  teeth.  It  is  better  to 
let  the  decalcification  proceed  slowly  for  a  longer  time  in  an  abundance 
of  fluid  changed  often,  in  order  that  the  carbon  dioxid  may  not  be 


64 

formed  too  fast,  accumulate  in  the  tissues,  inflate  and  distort  them. 
Many  fixers  contain  acid  (e.  g.,  see  §  §  12,  14,  17,  etc.),  and  in  their 
action  give  decalcification  enough  in  the  case  of  small  calcified 
objects. 

§  185.  Directions  for  use.  [10.]  The  tissue  to  be  decalcified 
had  best  be  first  thoroughly  fixed  and  hardened  by  one  of  the  approved 
methods,  and  should  be  in  82%  alcohol.  In  fixing,  structures  not 
needed  should  be  removed, — muscles  trimmed  away  from  the  bone, 
etc.  Bones  or  teeth  should  be  opened  with  nippers  or  a  saw,  so  that 
the  fluid  may  reach  the  marrow  or  pulp  cavity. 

Place  the  hardened  tissue  in  the  decalcifier,  where  it  should 
remain  until  the  lime  salts  have  been  entirely  removed,  as  may  be 
ascertained  by  inserting  a  fine  needle ;  if  any  calcified  matter  remains 
there  will  be  a  gritty  feeling  on  using  the  needle.  The  time  neces- 
sary for  complete  decalcification  will  depend  upon  the  size  and  den- 
sity of  the  calcified  tissue,  and  will  vary  from  3  to  15  days  or  longer. 
The  decalcifier  should  be  changed  after  the  first  day,  and  if  the  tis- 
sue is  large  it  is  best  to  change  it  subsequently  two,  three  or  more 
times  at  intervals  of  several  days. 

When  decalcification  is  complete  rinse  the  tissue  well  in  water  or 
67%  alcohol  for  a  few  minutes  and  place  it  in  67%  alcohol  for  one  or 
two  days  and  then  in  82%  alcohol  for  several  days,  or  until  ready  to 
imbed.  The  82%  alcohol  should  be  changed  once  or  twice  in  order 
that  the  nitric  acid  may  be  well  wrashed  out.  Although  paraffin  in 
many  cases  may  be  employed  for  imbedding,  the  celloidin  method  is 
generally  more  satisfactory. 

Hematoxylin  with  eosin,  hematoxylin  with  picro-fuchsin,  and 
hematoxylin  with  picro-carmine  afford  good  stains;  by  staining 
thoroughly  with  hematoxylin  a  differential  staining  of  bone  and 
cartilage  may  be  obtained.  Mallory's  connective  tissue  stain  fre- 
quently gives  interesting  pictures. 

(B).     Sections  of  Dry  Bone  or   Tooth. 

§  186.  Though  the  general  structure  of  bone  and  tooth  is  shown 
moderately  well  when  the  tissue  has  been  decalcified  (§  184).  the 
Haversian  canals,  canaliculi  and  lacunae  of  bone  and  the  dentinal 
tubules  of  the  teeth  are  shown  much  better  in  sections  of  dried, 
non-decalcified,  tissue  rendered  sufficiently  thin  for  microscopic  ex- 
amination by  grinding  or  filing. 


§  187.  Directions  for  procedure.  Prepare  thin  transverse  sec- 
tions of  dried  bone  in  accordance  with  the  directions  below.  Longi- 
tudinal (radial)  sections  and  tangential  (surface)  sections  may  also 
be  prepared  in  the  same  manner,  the  former  to  show  the  Haversian 
canals  and  their  anastomoses,  the  latter  to  indicate  the  shape  of  the 
lacunae  as  seen  in  a  different  plane. 

1.  Sawing  the  section.     Make  an  exact  transection  of  a  part  of 
the  shaft  of  a  long  bone.     The  section  should  be  about  1  cm.  long 
and  include  the  thickness  of  the  shaft  from  the  surface  to  the  medul- 
lary cavity.     Make  the  sections  about  1  mm.  thick. 

2.  Grinding  the  sections.     Place  the  piece  of  bone  on  a  cork  or 
piece  of  soft  wood  and  wet  it  with  water.     File  it  on  one  side  until 
smooth  and  then  turn  it  over.     Continue  the  filing  till  the  piece  is 
from  .05  to  .10  mm.  thick,  using  the  cover-glass  measurer  to  deter- 
mine the  thickness.     In  the  beginning  one  can  press  quite  hard  in 
filing;    as  the  section  thins,  more  care  should  be  exercised  and  the 
pressure  should  lessen. 

A  grinder,  such  as  a  fine  carborundum  wheel  or  emory  wheel  con- 
nected with  a  variable  speed  electric  motor  is  very  useful  and  greatly 
expedites  the  preparation  of  the  sections.  The  carborundum  wheel 
should  be  horizontal  and  the  sections  ground  on  the  flat  surface  of  the 
wheel,  water  being  used  to  carry  away  the  bone  dust. 

3.  Washing  and  drying  the  section.     When  the  section  is  thin 
enough,  rinse  it  and  dry  it  with  lens  paper. 

4.  Mounting  the  sections  in  hard  balsam.     To  prepare  the  bal- 
sam, put  two  or  three  large  drops  on  the  middle  of  a  slide  and  heat 
the  slide  in  some  way  to  drive  off  the  volatile  constituents.     Do  not 
heat  the  balsam  hot  enough  to  produce  bubbles.     When  the  balsam 
chips  after  cooling,  it  is  ready  for  use. 

In  mounting,  have  the  section  and  a  clean  cover  so  placed  that 
they  may  be  easily  and  quickly  grasped.  A  cork  somewhat  smaller 
than  the  cover-glass  should  be  within  reach,  and  also  a  stone  or 
piece  of  glass  upon  which  to  quickly  cool  the  specimen  as  soon  as  it 
is  mounted. 

Heat  the  slide  until  the  balsam  is  well  melted.  Put  the  slide 
upon  a  piece  of  paper,  grasp  the  piece  of  bone  with  the  forceps  and 
plunge  it  into  the  melted  balsam;  put  on  the  cover  as  quickly  as 
possible  and  press  it  down  with  the  cork;  finally  put  the  slide  on 
the  stone  or  glass  to  cool  the  balsam  quickly.  All  of  this  should  be 


66 

done  as  rapidly  as  possible,  and  if  done  rapidly,  the  air  will  be  retained 
in  the  lacunae  and  canaliculi,  and  cause  them  to  stand  out  as  black 
spots  and  lines.  If  soft  balsam  were  used  it  would  soon  drive  out 
the  air,  and  being  of  nearly  the  refractive  index  of  bone,  it  would 
obliterate  the  lacunae  and  canaliculi.  Further,  if  the  hot  balsam 
were  not  cooled  quickly,  the  air  would  be  driven  out  and  balsam 
would  take  its  place  in  the  spaces. 

MUSCLE. 

§  188.  Fresh.  Much  of  the  investigational  work  on  muscle 
has  been  done  on  fresh  muscle,  or  frozen  sections.  For  examination 
fresh  it  is  advantageous  to  have  very  thin  muscles.  Of  the  several 
muscles  that  have  been  recommended,  one  of  the  most  available  is 
the  M.  cutaneus  pectoris  of  the  frog.  This  may  be  prepared  by 
cutting  the  skin  in  the  midventral  line,  cutting  at  right  angles  to 
the  first  cut  across  to  the  angle  of  the  jaw,  thence  caudally  parallel 
to  the  first  cut.  The  skin  flap  so  formed  contains  the  insertion  of 
the  muscle  which  may  now  be  easily  dissected  free  and  removed  to- 
gether with  some  of  the  tissue  at  insertion  and  origin  to  handle  it  by. 
It  may  be  used  for  examination  fresh,  with  the  polarization  micro- 
scope, etc. 

§  189.  Isolation  Methods.  Nitric  acid  (§  44)  may  be  used  for 
plain  muscle  and  for  skeletal  muscle.  Potassium  hydroxid  (§  45) 
is  suitable  for  heart  muscle. 

(a)  Nitric  acid.  Place  in  the  nitric  acid  dissociator  the  fresh 
striated  muscle,  gland  or  organ  containing  the  muscle, — (plain 
or  striated,) — that  it  is  desired  to  isolate.  If  it  is  the  intention 
to  w^ork  out  the  anatomy  of  the  muscle  or  the  relation  of  the  muscular 
coats  in  an  organ,  the  entire  muscle  or  organ  should  be  taken;  other- 
wise, portions  will  suffice.  At  the  ordinary  temperature  of  the 
laboratory  the  dissociating  action  will  have  been  sufficient  in  from 
1  to  3  days;  test  at  intervals  with  needles  to  ascertain  whether 
the  fascicles  and  fibers  can  be  easily  separated;  or  fragments  may 
be  shaken  in  a  test  tube  or  vial  with  water  in  order  to  separate  the 
fibers. 

When  the  dissociation  is  sufficient  pour  off  the  acid  and  wash 
the  muscle  gently  but  thoroughly  with  water.  If  the  tissue  is  to  be 
stained  with  hematoxylin  or  carmine,  or  kept  for  any  length  of  time, 
drain  off  the  water  and  add  a  half -saturated  solution  of  alum.  For 


67 

permanent  storage,  pour  off  the  alum  solution  and  place  successively 
in  67%  and  82%  alcohol. 

For  temporary  examination,  tease  out  a  portion  of  the  muscle  in 
water,  separating  the  fibers  carefully  by  means  of  needles ;  cover  and 
examine. 

Permanent  preparations,  (a)  unstained.  After  teasing  out  with 
the  needles  drain  off  the  water  and  add  a  small  drop  of  glycerin 
or  glycerin-jelly;  cover,  and  seal  after  first  properly  cleaning  (§  §  162, 
163) .  (b)  stained. — Either  employ  the  nitric  acid  method  given  above 
or  (better;  Badertscher)  dissociate  at  incubator  temperature  (about 
38°  C.)  in  a  sat.  solution  of  mercuric  chlroid  with  10%  of  strong  nitric 
acid  added.  Test  at  intervals  and  when  dissociation  has  proceeded 
far  enough,  wash  out  the  dissociator  with  water  and  67%  alcohol. 
Stain  with  carmine  or  hematoxylin.  Mount  in  glycerin  (§  154-), 
glycerin -jelly  (§  155),  or  dehydrate,  clear  and  place  in  dilute  balsam 
(§  157-  Cf.  §  215). 

(b)  Potassium  hydroxid.  Place  in  the  fluid  small  pieces  of 
the  heart  muscle  of  a  fetal,  new-born  or  young  animal;  after  10  or  15 
minutes,  the  tissue  should  be  tested  with  needles  at  intervals  of  about 
five  minutes,  so  that  the  action  may  not  be  too  prolonged;  probably 
15  to  30  minutes  will  suffice.  As  soon  as  the  elements  separate 
readily,  pour  off  the  caustic  potash  solution  and  add  an  abundance 
of  60%  solution  of  potassium  acetate  (potassium  acetate,  60  grams; 
distilled  water,  40  c.  c.).  Take  small  fragments  and  tease  them  in 
this  solution,  or  shake  them  in  a  vial,  until  the  'cells'  are  separated 
from  each  other. 

For  temporary  examination,  cover,  in  a  drop  of  the  potassium 
acetate  solution.  For  permanent  preparations,  drain  off  the  potas- 
sium acetate  solution  and  add  a  small  drop  of  glycerin  or  glycerin- 
jelly. 

Stained  preparations.  Pour  off  the  potassium  acetate  solution 
and  add  a  half  saturated  solution  of  alum,  letting  it  remain  for  24 
hours  or  longer.  Tease  in  water,  stain  with  hematoxylin  or  carmine, 
wash  away  the  stain  with  water,  and  add  a  drop  of  glycerin  or  gly- 
cerin-jelly. Cover  and  seal  (§  162 — ). 

If  a  large  amount  is  desired,  the  tissue  may  be  carried  through 
the  various  steps  in  a  vial. 

§  190.  Sections.  To  bring  out  the  structure  of  the  fibrillae 
picro-aceto-formol  (§  17)  or  an  alcoholic  fixer  (§  §  23,  24)  is  preferable 


68 

although  the  sarcoplasm  is  not  so  well  preserved.  The  muscle  should 
be  moderately  distended  upon  cork,  before  fixing,  the  ends  secured 
by  small  pins.  10%  formalin  or  Zenker's  fluid  or  Mercuric  chlorid 
may  also  be  used,  the  sarcoplasm  being  much  better  fixed  in  di- 
chromate,  osmic  acid  or  formalin  mixtures.  Iron  hematoxylin  is 
particularly  indicated  as  a  stain  for  muscle  (§  94).  Mallory's  con- 
nective tissue  stain  (§  120)  is  found  to  give  an  excellent  differentiation 
(Kingery). 

To  differentiate  muscle  in  situ,  picrofuchsin  may  be  used,  which 
stains  muscle  yellow  or  orange,  the  surrounding  connective  tissue 
red.  Mallory's  connective  tissue  stain  is  also  frequently  useful. 

To  differentiate  the  intercalated  discs  of  cardiac  muscle,  fix  in 
mercuro-nitric  mixture  (§  20) ;  imbed  in  paraffin ;  stain  sections  with 
haemalum  (§  89),  diluted,  12  to  24  hours;  differentiate  with  acid 
alcohol;  dehydrate,  clear,  mount  in  balsam. 


THE   NERVOUS    SYSTEM. 

An  analytical  grouping  of  the  numerous  methods  used  in  the 
study  of  the  finer  structure  of  the  Central  Nervous  System  is  pre- 
mature. The  most  salient  point  is  the  prominent  part  that  reduc- 
tion processes  seem  to  play.  The  more  important  methods  here  pre- 
sented deal  (a)  with  the  finer  structure  of  cell  and  fiber; — demonstra- 
tion of  the  tigroid  substance  and  fibrillae ;  (b)  the  differential  staining 
of  the  myelinic  nerve  fiber  (Weigert  and  Marchi  methods);  (c)  the 
morphology  of  the  elements  (neurones)  as  revealed  by  the  chrome- 
silver  impregnation  methods  (Golgi  methods)  or  the  use  of  intra- 
vitam  (methylene  blue)  methods. 

§  191.  Isolation  of  Nerve  Cells.  Employ  formaldehyde  disso- 
ciator  for  the  isolation  of  the  nerve  cells  of  the  spinal  cord  and  of 
the  cerebral  cortex,  proceeding  as  follows : 

Split  the  spinal  cord  along  its  median  plane,  separating  thus 
the  two  halves,  and  place  it  in  an  abundance  of  the  dissociating 
fluid.  The  cerebral  cortex  should  be  cut  into  small  pieces  by  sec- 
tions vertical  to  the  surface.  Allow  it  to  remain  in  the  dissociator 
from  2  to  24  hours;  for  the  best  results  a  stay  in  the  fluid  of  more 
than  24  hours  is  not  so  satisfactory;  although  isolated  cells  are 
readily  obtained  their  processes  are  broken  off  much  nearer  the  cell 
body. 


Place  a  fragment  of  the  gray  matter  of  the  spinal  cord  or  the 
cortex  of  the  cerebrum  on  a  clean  slide  in  a  drop  of  J^%  Congo  Red 
(§  113)  or  1/10%  eosin  in  formaldehyde  dissociator;  with  the  blade 
of  a  scalpel  crush  the  tissue,  grinding  it  thoroughly  with  a  rotary 
movement,  which  will  reduce  it  to  small  pieces.  Gather  the  debris, 
drain  off  the  fluid,  and  add  a  drop  of  glycerin  containing  stain. 
Cover  and  examine,  tapping  the  cover  sharply  with  the  handle  of  the 
scalpel  to  shake  out  the  processes  of  the  cells  and  free  them  from 
surrounding  matter.  Examine,  searching  for  cells  with  many  and 
long  processes,  and  if  a  satisfactory  preparation,  seal  according  to 
§  119. 

§  192.  Isolation  of  Nerve  Fibers,  (a)  For  isolation  of  myel- 
inic  nerve  fibers,  with  the  preservation  and  blackening  of  the  myelin, 
and  for  amyelinic  fibers,  employ  osmic  acid  dissociator  (§  42). 

(b)  For  the  isolation  of  myelinic  nerve  fibers  with  the  removal 
of  the  myelin  for  the  demonstration  of  the  axis  cylinder,  neuro- 
lemma  and  framework  of  the  sheath,  fix  nerves  in  Dichromate- 
acetic  or  similar  fluid  one  or  more  days,  wash  in  water,  pass  up 
through  the  alcohols,  dehydrate,  remove  the  myelin  by  placing  the 
tissue  in  a  fat  solvent, — chloroform,  for  one  or  more  days,  95%  alco- 
hol 1  day,  pass  through  the  alcohols  to  water,  stain  in  Delafield's 
hematoxylin  12  to  24  hours,  wash  in  water,  pass  up  through  the 
alcohols,  dehydrate  and  place  in  clearer.  Out  of  this  the  small  bun- 
dles of  the  nerve  fibers  may  be  teased  apart  with  needles,  care  being 
taken  to  keep  the  fibers  as  nearly  parallel  as  possible.  Mount  in 
balsam. 

§  193.  Gold  Chlorid  Methods.  These  methods,  which  are 
widely  serviceable,  depend  upon  the  reduction  of  gold  chlorid  solu- 
tions by  the  tissues  through  the  agency  of  (a)  sunlight  or  (b)  various 
chemical  substances  of  which  the  acids,  formic,  acetic,  citric,  etc., 
are  particularly  used.  Either  one  or  both  of  these  agencies  may  be 
used.  Usually  fresh  tissue  is  used  although  the  method  may  be 
applied  to  fixed  tissue,  particularly  as  a  neurofibrilla  stain  [6,  30]. 
It  is  the  method  par  excellence  for  staining  motor  nerve  terminations 
for  which  purpose  the  following  method  is  serviceable. 

§  194.  [21].  Fresh  tissue  or  (better)  tissue  fixed  in  10%  formalin  may  be 
used.  Place  small  pieces  of  muscle  containing  the  endings  for  30  minutes  in  10% 
formic  acid  solution.  Remove  to  1%  gold  chlorid  for  30  to  40  minutes,  avoiding 
direct  sunlight;  the  tissue  becomes  yellow.  Transfer  again  to  a  2%  formic  acid 
solution  in  which  the  tissue  should  remain  for  1  or  2  days  in  the  dark  (rich  purple 


70 

color).  A  bluish-purple  indicates  too  great  a  reduction.  Wash  in  distilled  water 
for  an  hour  or  so. 

If  it  is  desired  to  make  a  teased  preparation,  transfer  to  glycerin  in  which 
the  fibers  may  be  cautiously  teased  apart,  taking  care  not  to  separate  them  too 
much.  Permanent  mounts  may  be  made  in  glycerin  or  glycerin-jelly. 

If  sections  are  called  for,  dehydrate,  clear  (xylene  method,  §  52),  and  imbed  in 
paraffin.  Celloidin  may  also  be  used. 

With  fresh  tissue  the  treatment  by  formic  acid  may  cause  too 
marked  a  swelling  and  distortion ;  in  which  case  the  Ranvier  method 
[30]  may  preferably  be  used. 

§  195.  Tigroid  substance  (Nissl's  bodies).  The  stainable  sub- 
stance in  the  cell  body  of  nerve  cells  resembles  the  chromatin  of 
the  nucleus  in  its  reactions,  staining  with  basic  stains  of  which  a 
number  are  suitable.  Alcohol,  Carnoy's  fluid  (§  26,  27),  mercuric 
chlorid,  or  formalin  may  be  used  as  fixers,  preferably  one  of  the  first 
three.  Methylene  blue  or  toluidin  blue  are  the  usual  stains. 

§  196.  NissPs  Method  (modified).  Imbed  in  paraffin  or  cel- 
loidin  tissue  that  has  been  fixed  in  95%  alcohol  or  Carnoy's  fluid; 
cut  the  sections  rather  thick,  15  to  20^.  The  sections  may  either  be 
fastened  to  the  slide  or  carried  on  as  free  sections.  Stain  the  sec- 
tions in  a  1%  aqueous  solution  of  methylene  blue  or  toluidin  blue 
for  5  to  10  minutes,  heating  it  until  it  steams.  Permit  it  to  cool, 
rinse  in  water,  dehydrate  and  differentiate  in  absolute  alcohol,  clear 
with  oil  of  cajuput  and  xylene  and  mount  in  xylene  balsam.  The 
nerve  cells  and  nuclei  will  be  stained  blue,  all  else  colorless.  In  the 
cell-bodies  of  the  nerve  cells,  the  tigroid  substance  will  be  stained. 
Should  the  stain  not  be  selective  enough,  differentiate  for  a  few 
seconds  before  dehydrating  with  a  mixture  of  anilin  1  part,  95% 
alcohol  9  parts. 

If  celloidin  is  used  for  imbedding,  it  should  be  remembered  that 
it  should  be  dissolved  away  in  the  differentiation  (absolute  alcohol). 

§  197.  H eld's  Method  (modified).  Fix  in  mercuric  chlorid 
(or  as  for  Nissl's  method),  imbed  in  paraffin,  cut  sections  5  to  10^ 
and  stain  15  minutes  in  1%  solution  of  erythrosin  in  67%  alcohol, 
rinse,  stain  10  minutes  in  1%  aqueous  solution  of  toluidin  blue,  dif- 
ferentiate briefly  in  1 /10th%  alum  solution,  dehydrate  rapidly  in 
95%  and  absolute  alcohol,  clear  in  xylene,  mount  in  balsam.  Alka- 
line methylene  blue  or  Nissl's  soap  solution  (y&%  methylene  blue  in 
1/5%  soap  solution)  may  be  used  in  either  of  these  two  methods  if 
desired. 


71 

§  198.  Neurofibrillae  (Simarro-Cajal  Methods).  Three  of  Ca- 
jal's  methods  may  be  given :  Formula  3a.  1 .  Fixation  in  ammonia- 
cal  alcohol  (2  to  10,  usually  4  to  5,  drops  of  ammonia  per  50  c.  c.  of 
95%  alcohol),  20  to  48  hours;  2.  Mop  up  with  absorbent  paper  and 
3.  Place  in  !>£%  silver  nitrate  solution  for  4  to  5  days  at  32  to  40°  C. ; 
the  tissue  when  ripe  should  be  light  gray;  4.  Wash  for  a  few  minutes 
in  distilled  water;  5.  Reduce  in  a  solution  of  1  to  2  grams  hydro- 
chinon  or  py rogallol ;  water,  100  c.  c.;  formalin,  5  c.  c.;  for  24  hours; 
6.  Wash  in  wTater;  7.  Imbed  by  the  paraffin  method;  8.  Section 
and  mount  in  balsam  or  damar.  Recommended  for  spinal  cord, 
cerebellum,  spinal  ganglia. 

Formula  l^a.  1.  Fix  small  pieces  of  tissue  (5  mm.  thick  or  less) 
for  6  to  12  hours  in  formalin,  15  c.  c.,  water  85  c.c.;  2.  Wash  6 
hours  or  longer  in  running  water;  3.  Place  for  24  hours  in  50  c.  c. 
of  alcohol  with  5  drops  of  ammonia  added;  4.  Mop  with  absorbent 
paper;  5.  Place  in  1^%  silver  nitrate  solution  for  4  to  5  days  (35 
to  38°  C.);  The  remaining  steps  as  in  formula  3a.  Recommended 
for  sympathetic  ganglia,  cerebrum,  cerebellum. 

Formula  5a.  1.  Fix  small  pieces  for  6  to  8  hours  in  water  and 
pyridin,  each  equal  parts;  then  for  18  to  24  hours  in  pure 
pyridin;  2.  Wash  for  several  hours  in  running  water;  3.  Place 
in  90%  alcohol  for  1  day ;  4.  Mop  with  absorbent  paper;  5.  Place 
in  \Yi%  silver  nitrate  solution  for  4  to  5  days  (35  to  38°  C.);  The 
remaining  steps  as  in  3a  and  4a.  Recommended  for  embryonic  and 
fetal  tissue  (neurogenesis),  regeneration,  cerebrum. 

By  these  "photographic"  methods,  fibrillae,  fibrillar  networks, 
changes  in  histogenesis,  etc.,  may  be  demonstrated. 

For  other  methods  of  demonstrating  the  neurofibrillae, — Biel- 
schowsky's,  Bethe's  toluidin  blue  method,  Apathy's  hematein 
method,  etc.,  consult  larger  wwks  on  technique  [6,  30], 

§  199.  The  Weigert  Method  for  staining  differentially  the 
myelin  of  myelinic  nerve  fibers.  This  method  in  all  its  various 
forms,  depends  upon  the  power  of  the  myelin,  probably  through  the 
reducing  fatty  acid  present,  to  combine  writh  and  hold  in  (nearly) 
insoluble  form  the  chromium  (oxid),  which  thus  serves  as  a  pri- 
mary mordant  for  a  copper  or  iron  hematoxylin  stain,  which  is  sub- 
sequently differentiated  by  an  oxidizer  as  a  bleacher.  The  important 
steps  are: — (1)  fixing  and  mordanting  in  dichromate  solutions;  usually 
the  potassium  salt  is  chosen;  (2)  a  second  mordantage  in  copper 
(acetate),  (3)  the  staining;  (4)  the  differentiation.  The  point  at 
which  the  imbedding  and  sectioning  are  introduced  is  of  secondary 


72 

importance.  It  should  be  remembered,  however,  that  the  fatty  sub- 
stances (lipoids)  of  the  myelin  upon  which  the  method  depends  are 
soluble  in  the  reagents  of  both  the  paraffin  and  cello idin  methods,  less 
so  in  the  latter — and  in  acids,  and  that  even  the  dichromate  mordan- 
tage  does  not  preserve  them  perfectly.  The  dichromate  mordant  age 
must  thus  be  given  before  the  imbedding  is  begun  (alcohols) ,  and  pref- 
erably with  the  fresh  tissue.  Practically  the  only  fixer  that  is  indif- 
ferent in  this  respect  and  after  which  the  dichromate  may  first  be  used 
is  formalin.  Other  dichromate  fixers  such  as  Zenker's  fluid  with  the 
acetic  acid  reduced  to  about  1/5%  may  be  used,  but  it  is  well  to  let 
them  act  only  a  relatively  short  time,  and  continue  the  mordantage 
with  simple  dichromate  solutions.  Aside  from  this  the  point  at  which 
the  imbedding  and  sectioning  are  introduced  is  of  secondary  im- 
portance; thus,  Strong  [41]  combines  the  copper  and  chromium 
mordantage  by  using  copper  dichromate;  the  former  may  also  follow 
the  dichromate  treatment  before  imbedding  is  begun,  in  case  of 
celloidin  imbedding  it  may  be  applied  to  the  celloidin  imbedded 
block,  or  after  the  sections  are  cut.  Street er  [40]  stains  (in  toto,) 
4  to  6  days  as  well  as  mordants  before  the  imbedding.  Weigert  has 
added  to  both  the  primary  and  secondary  mordants  chromium 
fluorid  to  (1)  hasten  the  process  and  (2)  prevent  precipitates;  the 
formulas  are:  (a)  5%  potassium  dichromate,  100  c.  c.;  chromium 
fluorid,  2  grams;  (b)  5%  copper  acetate,  100  c.  c.;  chromium  fluorid, 
2  grams;  glacial  acetic  acid,  4  c.  c.  (b)  is  especially  indicated  if  the 
secondary  mordantage  is  given  before  the  sectioning.  Whatever 
modification  of  the  method  is  employed,  the  reduction  of  the  dichro- 
mate by  the  tissue  in  the  primary  mordantage  should  fully  reach 
the  dark  brown  stage,  but  not  pass  it  (i.e.,  become  green).  Sheldon 
[4]  gives  a  good  resume  of  the  method.  The  following  method  is 
serviceable : — 

1.  Fix  tissue  for  1  to  2  days  in  Zenker's  (see  above)  Orth's,  or 
Kelly's  fluid,  10%  formalin,  Muller's  fluid,  or  potassium  dichromate 
solution. — 

2.  Mordant  until  dark  brown  in  3%  and  5%  aqueous  solution 
of  potassium  dichromate.     This  usually  takes  about  4  weeks;    or, 
shorten  the  period  by  using  the  dichrornate-chromium  fluorid  mix- 
ture (above)  when  about  5  days  should  suffice ; 

3.  Wash  in  running  water  1  or  2  days,  and 

4.  Pass  up  through  the  alcohols,  preferably  keeping  the  tissue 
in  the  dark. 


73 

5.  Imbed  in  celloidin  or  paraffin. 

6.  Stain  and  differentiate  sections  by  the  Weigert  copper  hema- 
toxylin  method  (§  95). 

7.  Mount  in  neutral  or  alkaline  balsam. 

Large  sections  are  usually  best  carried  on  as  free  sections.  The 
differentiation  of  the  stain  should  be  carefully  watched  and  stopped 
when  the  fibers  are  a  rich  dark  blue  on  a  yellow-brown-background. 
The  reagents  used  in  dehydrating,  clearing  and  mounting  should 
be  neutral  or  alkaline, — not  acid. 

§  200.  Pal's  method  may  be  used  if  it  is  desired  to  stain  the 
nerve  cells  subsequently. 

Fix  and  mordant,  in  the  dichromate  as  above;  omit  the  copper 
mordantage;  imbed  and  section,  staining  the  sections  with  the  strong 
hematoxylin  used  in  the  Weigert  method  (§  199)  until  the  sections  are 
a  blue-black. 

Rinse  the  sections  in  tap  water  and  differentiate  by  treating  for 
a  short  time  (20  to  30  seconds)  with  a  1  /10%  aqueous  solution  of 
potassium  permanganate  and  for  a  few  seconds  with  a  mixture  of 
1%  oxalic  acid  and  1%  potassium  sulphite,  equal  parts.  The  action 
will  be  very  rapid  and  must  be  carefully  watched.  Wash  the  sec- 
tions %  hour  in  running  water.  *  Counter-stain  with  a  red  stain 
(eosin,  erythrosin,  carmine,  etc.)  if  desired. 

§  201.  Marchi  Method.  This  method  of  staining  differen- 
tially degenerating  myelinic  nerve  fibers  depends  upon  the  fact  that 
potassium  dichromate  (or  chromic  acid)  is  able  to  satisfy  the  re- 
ducing power  of  myelin  but  does  not  oxidize  the  globules  of  free 
fatty  acid  (?)  formed  in  the  degeneration  of  the  myelinic  sheath 
of  the  fiber,  which  may  be  subsequently  blackened  by  the  reduction 
of  osmium  tetroxid.  Important  points  in  the  successful  application 
of  the  method  are:  (a)  the  length  of  time  the  degeneration  should 
be  allowed  to  proceed  before  treating  with  potassium  dichromate, 
(b)  the  time  in  the  dichromate  mordant,  (c)  the  time  in  the  osmic 
acid  mixture  (sufficient  and  complete  penetration),  (d)  the  preser- 
vation in  situ  and  final  mounting  of  the  osmicated  fat  granules;  the 
difficulties  here  are  those  of  fat  preservation  in  general  (§  224). 

(a)  The   optimum   will    vary   and   must   often   be   determined 
experimentally:     in  general, — for  cold  blooded  animals;    (Toad),  30 
to  40  days;    for  mammals,  12  days. 

(b)  8  to  10  days  in  Muller's  fluid  or  3%  potassium  dichromate 
is  usually  enough;    a  longer  time  does  no  harm. 


74 

(c)  The  osmic  acid  is  useful  in  1%  aqueous  solution  usually 
mixed  with  a  potassium  dichromate  solution.     6  to  10  days  suffice;  a 
longer  time  does  no  harm  (brittleness) . 

(d)  See  below. 

§  202.     As  employed  by  van  Gehuchten  [18] : — 

1.  Harden  in  3%  potassium  dichromate  solution  for  3  weeks; 

2.  Transfer  to  a  mixture  of  1%  osmic  acid  solution,   1  part; 
3%  potassium  dichromate,  4  parts;    for  3  weeks,  blocks  of  tissue 
not  more  than  2  mm.  thick.     Use  abundance  of  the  fluid  and  change 
2  to  3  times  if  deemed  necessary. 

3.  Wash  in  running  water  for  12  to  24  hours. 

Avoid  the  paraffin  and  celloidin  imbedding  methods  if  pos- 
sible. Of  these  two  the  celloidin  method  is  preferable.  For  further 
treatment,  see  §  227. 

§  203.  Flemming's  or  Benda's  fluids  (§  §  19,  227)  may  be  used 
instead  of  the  Marchi  method  for  the  same  purpose  with  small  objects, 
and  peripheral  nerves,  etc. 

§  204.  The  Golgi  Methods,  whose  field  of  application  is  not 
confined  to  the  nervous  system  (gland  ducts,  bile  capillaries,  blood 
capillaries,  secretory  canaliculi,  muscle,  etc.)  consist  in  (a)  mordant- 
ing the  fresh  or  living  tissue  for  a  sufficient  length  of  time  in  a  dichro- 
mate solution,  usually  containing  as  well  osmic  acid  or  formalin, 
and  then — (b)  transferring  to  a  silver  nitrate  solution,  whereupon 
certain  of  the  nervous  elements  become  outlined  more  or  less  com- 
pletely by  impregnation  with  a  chrome-silver  combination. 

The  reaction  probably  depends  on  the  presence,  in  certain  "phy- 
siological states"  of  the  elements  of  a  substance  or  substances  which 
combines  with  the  chromium  salt  (with  reduction?)  and  through  it 
with  the  silver  salt.  These  hypothetical  substances, — or  possibly 
physical  states,— seem  to  disappear  more  or  less  rapidly  after  the 
death  of  the  animal  and  their  power  to  hold  the  silver  in  combination 
to  decrease  with  the  progress  of  the  dichromate  mordantage  beyond 
a  certain  point.  If  successful,  certain  of  the  cells  and  their  processes, 
— amyelinic,  and  to  a  certain  extent,  myelinic  nerve  fibers,  are  out- 
lined by  an  impregnation,  black  by  transmitted,  brown  by  reflected 
light. 

§  205.  The  method  is,  however,  capricious;  success  depends  on 
(a)  the  kind  of  animal;  different  parts  and  tissues  react  more  satis- 
factorily in  some  animals  or  classes  of  animals  than  in  others,  (b) 


75 

The  age  of  the  animal;  some  regions  of  the  nervous  system  give  bet- 
ter results  in  young  or  fetal  animals ;  other  parts  take  the  stain 
better  in  older  animals,  etc.  (c)  The  time  of  mordantage;  it  is  neces- 
sary that  the  tissue  be  mordanted  a  certain  length  of  time,  constant 
(relatively)  for  a  certain  kind  of  tissue  under  the  conditions  above 
(a  and  b).  It  is  necessary  that  the  best  amount  of  dichromate  mor- 
dantage be  given,  (d)  Different  organs  and  regions  of  the  central 
nervous  system  vary  greatly  in  the  ease  with  which  they  can  be  made 
to  furnish  satisfactory  impregnations.  Almost  certain  impregna- 
tions of  hippocamp  can  be  gained;  cerebral  cortex  is  likewise  quite 
easy  to  stain.  With  the  olfactory  bulb  the  action  is  not  constant 
though  fairly  complete.  The  optic  lobes  and  retina  of  birds  and 
large  reptiles  are  more  satisfactory  than  those  of  mammals.  The 
my  el  (spinal  cord)  of  embryo  birds  (7  to  14  day  chick  best)  is  gen- 
erally more  satisfactory  than  that  of  mammals;  in  any  case,  fetal  or 
new-born  animals  should  be  employed.  Difficult  are  satisfactory 
impregnations  of  sympathetic  ganglia,  organs  of  special  sense  and 
the  intrinsic  nerves  of  the  viscera. 

The  important  forms  of  the  method  are: — (a)  the  Slow  Method; 
mordantage  in  dichromate  solutions  of  preferably  increasing 
strength,  2,  3,  5%,  for  1  to  4  months  depending  on  the  temperature, 
strength  of  solution,  etc.  (b)  The  Rapid  Method;  in  this  another 
oxidizer,  osmic  acid,  is  combined  with  the  dichromate  with  a  reduc- 
tion in  the  duration  of  the  treatment  to  a  few  days.  Combination  of 
(a)  and  (b)  are  sometimes  serviceable;  (c)  Double  (or  triple)  Im- 
pregnations, obtained  by  repeating  (b).  Important  modifications 
are:  (1)  substitution  of  formalin  for  the  osmic  acid  in  (6),  (2) 
mercuric  chlorid  instead  of  silver  nitrate  (Cox's  Method).  The 
Golgi  methods  have  been  widely  applied  and  for  details  the  individual 
papers  may  be  consulted  [6]. 

§  206.  Golgi's  Rapid  Method.  This  is  the  most  generally 
serviceable  of  the  different  methods. 

Directions  jor  use.  Tissue  of  a  (preferably)  yoiing  animal  is 
placed  in  a  mixture  of  4  parts  of  3%  potassium  dichromate  and  1 
part  of  1%  osmic  acid.  The  amount  of  the  fluid  should  be  at  least 
twenty  times  the  bulk  of  the  tissue  and  should  be  changed  as  soon 
as  it  grows  turbid  or  loses  the  strong  characteristic  odor  of  the  osmic 
acid. 

After  the  action  has  proceeded  to  the  right  degree  (§  207),  rinse 
the  tissue  in  water  for  about  5  minutes  and  place  for  15  minutes  in 


76 

a  ]/4C/c  solution  of  silver  nitrate,  and  then  for  2  or  more  days  in  a 
%%  solution  of  silver  nitrate,  preferably  keeping  it  in  the  dark. 
Without  washing,  imbed  rapidly  in  celloidin  as  follows: 
(a)     Dehydrate  2  to  3  hours  in  95%  alcohol,  changed  two  or 
three  times;    (b)  place  in  thin  celloidin  for  20  minutes,  in  thick  cel- 
loidin for  20  to  30  minutes;    (c)  imbed  in  thick  celloidin,  on  a  block 
of  wood  (best  ;  §  65a) ;    (d)  harden  the  mass  in  chloroform  for  20  to 
30  minutes,  and  (e)  place  the  block  in  clarifier  and  cut,  sections 
being  50  to  100  ^  thick  according  to  the  nature  of  the  tissue  and  the 
character  .of  the  impregnation. 

(/)  Place  the  sections  in  95%  alcohol  for  a  few  minutes;  clear 
in  carbol-xylene  and  mount  in  balsam  by  placing  the  section  on  the 
slide,  absorbing  the  clearer  thoroughly  by  means  of  tissue  paper  and 
spreading  over  it  thick  xylene  balsam.  Do  not  cover.  Later,  when 
the  balsam  has  hardened  somewhat,  it  may  be  melted  by  heat  and 
much  of  the  superfluous  balsam  drained  from  the  section  and  scraped 
away  with  a  knife,  and  a  cover  glass  added  if  desired. 

§  207.  Time  oj  hardening.  From  results  of  Cajal,  van  Gehuch- 
ten,  and  others,  and  from  general  laboratory  experience,  the  follow- 
ing periods  will  probably  be  found  approximately  correct.  In 
general:  The  best  results  are  to  be  obtained  with  kittens  3  to  20 
days  old,  puppies  2  weeks  old,  rats  8  to  10  days,  rabbits  8  days,  (a) 
For  cerebral  cortex  (and  hippocamp) :  New-born  kitten,  1  to  2  days; 
kitten  half  grown  (3  to  4  months),  3  to  4  days;  new-born  rabbit,  24 
hours;  rabbit  one  month  old,  2  to  3  days;  adult  mice,  3  to  4  days. 

(b)  For  spinal  cord :     Chick  of  5  to  6  days' incubation,  24  hours; 
chick,  14  to  15  days'  incubation,  3  days;   new-born  kitten  or  puppy, 

2  to  3  days.     Frog  tadpoles  (large)  3  to  5  days. 

(c)  Cerebellum:     New-born  kitten,   1   to   2  days;     kitten  half 
grown,  4  days. 

(d)  Sympathetic  system:     Chick  of  14  to  18  days'  incubation, 

3  days. 

(e)  Retina :     1  to  3  days. 

(/)     Olfactory  mucous  membrane:     3-4-7  days. 

§  208.  Intra  vitam  Methylene  Blue.  Methylene  blue  shares 
with  a  number  of  coal  tar  dyes  the  power  of  staining  during  life 
nerve  cells  and  fibers  and  certain  cytoplasmic  granules.  Like  the 
Golgi  methods,  "intra  vitam"  staining  of  nervous  tissue  is  capricious; 
applicable  only  to  living  or  fresh  tissue  and  depends  upon  unknown 


77 

substances  or  conditions  that  become  changed  after  death.  In  the 
reaction  reduction  of  the  methylene  blue  to  its  leucobase  (colorless) 
by  the  nervous  tissues  appears  to  play  an  important  part.  In  general, 
the  technique  involves:  (1)  Bringing  a  methylene  blue  solution  of 
sufficient  strength  in  contact  with  the  (essentially)  living  nervous 
elements,  (2)  permitting  it  to  remain  a  sufficient  length  of  time  for 
the  staining  reaction,  (3)  exposing  the  tissue  to  the  action  of  the 
oxygen  of  the  air  until  the  stain  is  fully  developed, — re-oxidation 
of  any  leuco-methylene  blue  and  satisfying  the  reducing  reaction  of 
the  tissue;  then — (4)  either  examining  at  once  or  fixing  the  stain 
in  situ  by  its  precipitation  in  an  insoluble  form  for  its  preservation 
(imbedding  and  sectioning) . 

1.  The  methylene  blue  may  be  brought  in  contact  with  the 
neurones  by  injection,   (a)  through  the  vascular  system, — aorta  if 
the  animal  is  small,  artery  supplying  the  part,  if  large; — (b)  into 
the  body  cavities ;   (c)  subcutaneously :  or,  by  immersion  of  the  organ 
or  part,  or  the  entire  animal  if  small  (many  invertebrates).     Keep- 
ing in  mind  the  end  result  desired,  the  best  method  will  suggest  itself 
in  a  particular  case.     Cajal  cut  parallel  slits  in  the  cerebral  cortex 
and  inserted  the  methylene  blue  in  powdered  form  or  as  a  saturated 
solution.     In  general,  the  solutions  should  be  as  dilute  as  possible; 
of  1/15  to  1/4%  strength  in  physiological  salt  solution,  the  more 
direct  the  application  the  weaker.     It  is  well  to  have  on  hand  a  1% 
stock  solution  in  physiological  salt  solution  and  dilute  it  (with  salt 
solution)  as  desired.     Of  the  methylene  blue  preparations  that  are 
available,  Ehrlich's  or  "B.  X."  are  more  generally  used.     A  combi- 
nation of  injection  and  immersion  is  often  advisable.     In  introduc- 
ing the  stain  by  injection,  first  remove  the  blood  by  washing  out  with 
physiological  salt  solution  or  by  bleeding  and  let  the  injection  be 
full,  i.  e.,  through  the  capillaries  into  the  veins.     If  a  mammal  is  be- 
ing dealt  with,  salt  solution  and  staining  solution  should  be  warmed 
to  body  temperature  (35  to  38°  C.). 

2.  It  is  difficult  to  give  any  general  rules  as  to  the  time  the 
methylene  blue  solution  should  remain  in  contact  with  the  tissue 
before  exposure  as  it  is  best  to  determine  it  experimentally  in  each 
case.     The  time  should  be  shorter  for  warm  blooded,  longer  for  cold 
blooded  animals.     If  introduced  by  injection,  20  to  30  minutes  for 
a  mammal,  2  to  12  hours  for  a  cold  blooded  form,  may  be  suggested; 
the  organ  or  part  should  then  be  removed,  wet  with  the  dilute  stain 
(perhaps  1  /15%  strength)  for  another  period  of  time, — ^  to  1  hour, 


78 

access  of  oxygen  to  the  point  desired  being  kept  in  mind.  At  inter- 
vals, free  hand  sections  should  be  examined  under  the  microscope  to 
determine  the  state  of  the  reaction.  If  the  tissue  is  mammalian,  it 
should  be  kept  protected  from  evaporation  during  this  time  and 
warm,  as  in  an  incubator. 

If  the  stain  was  applied  by  immersion,  a  shorter  time  suffices; — 
up  to  15  minutes  or  so  with  a  subsequent  exposure  to  the  air  of  l/2 
to  1  hour  wet  with  the  dilute  solution. 

Small  aquatic  animals  may  be  immersed  in  very  dilute  solu- 
tions (1/100  to  1/1,000%),  the  optimum  strength  and  time  of  im- 
mersion being  experimentally  determined. 

In  some  instances  (particularly  parasitic  worms)  the  tissue 
hold  the  methylene  blue  in  reduced  form  in  spite  of  exposure  to  the 
air  and  the  color  is  only  developed  when  placed  in  the  fixer  (below). 
In  any  event  it  is  better  to  fix  the  stain  earlier  than  later. 

4.  Two  methods  are  standard  for  preserving  tissue  stained 
intra  vitam  with  methylene  blue,  the  first  of  these  (Dogiel's)  is  suit- 
able only  for  such  tissue  as  may  be  exposed  for  study  by  teasing; 
the  second  (Bethe's)  may  be  used  both  for  such  preparations  and 
those  which  it  is  desired  to  imbed  and  section. 

Dogiel's  Method.  Immerse  the  tissue  in  a  saturated  solution  of 
ammonium  picrate  (orange-yellow  needles)  for  2  to  24  hours  according 
to  the  size  of  the  piece,  using  abundance  of  fluid.  If  maceration 
occurs,  Dogiel  suggests  addition  of  1%  of  1%  osmic  acid.  Transfer 
tissue  to  equal  parts  of  the  ammonium  picrate  solution  and  glycerin  in 
which  the  tissue  may  be  preserved,  teased,  and  mounted. 

Bethe's  Method.  Immerse  the  tissue  in  a  5  to  10%aqueous  solu- 
tion of  ammonium  molybdate  for  1  to  24  hours  according  to  the  size  of 
the  piece,  using  abundance  of  the  fluid.  Trim  the  tissue  as  desired, 
removing  all  unnecessary  parts,  dividing  it  into  smaller  pieces,  etc. 
Wash  in  distilled  water,  changed  several  times,  for  1  to  3  hours. 
Dehydrate  rapidly  in  95%  and  absolute  alcohol, — 4  to  6  hours, 
shortening  the  time  if  possible.  Imbed  rapidly  in  celloidin  (§  206) 
which  may  be  hardened  in  67%  alcohol  and  sections  cut. 

It  may  be  advisable,  particularly  in  summer  work,  to  have  the 
water  and  alcohol  specially  cooled  to  prevent  dissolving  of  the  stain 
in  the  alcohol.  Tissue  already  fixed  in  the  ammonium  picrate  may 
be  refixed  in  the  ammonium  molybdate  solution.  Indeed,  this 
double  fixation  is  recommended  by  Bethe  as  particularly  suitable  for 
invertebrate  material. 


79 

The  paraffin  method  is  not  advisable  if  the  above  method  suf- 
fices. If  it  is  desired,  however,  after  the  dehydration,  clear  thor- 
oughly with  oil  of  cloves  followed  by  xylene  and  infiltrate  in  paraffin 
(§  52).  In  treating  the  sections,  avoid  alcohol  as  much  as  possible. 

-Section  or  in  toto  staining  may  be  applied,  preferably  carmine 
(not  alkaline  or  acid  formulae) . 

This  important  method  has  been  elaborated  largely  through  the 
work  of  Bethe,  Cajal,  Dogiel,  Huber,  Retzius  and  others,  for  which 
consult  [6]. 

The  method  may  be  used  for  the  staining  of  Neurofibrillae ;  for 
its  applications  for  this  purpose,  consult  the  special  articles. 

§  209.  Neuroglia  Stain.  Tissue  is  fixed  for  24  hours  in  copper 
dichromate-sublimate-acetic  (1/5%)  mixture  (§  16)  and  subsequently 
mordanted  3  or  4  days  in  2.5%  copper  dichromate.  Imbed  in  paraffin, 
Sections  (5  to  10^)  are  fastened  to  the  slide  and  the  Benda  stain  is 
used,  as  follows: — (1)  4%  ferric  alum  for  24  hours;  rinse  well  in  dis- 
tilled water  and  (2)  place  for  24  hours  in  a  dilute  solution  (amber 
yellow)  of  sodium  sulphalizarinate  (concr.  sol'n.  in  70%  alcohol 
added  to  distilled  water).  Rinse  in  distilled  water,  blot  with  absorb- 
ent paper,  and  stain  (3)  in  a  1/10%  aq.  sol'n.  of  toluidine  blue, 
heating  it  until  it  steams,  cooling  and  staining  15  minutes.  (4)  Rinse 
with  distilled  water  and  treat  for  a  few  seconds  with  acid  alcohol  (70% 
alcohol,  100  c.  c.;  concr.  Hcl,  6  drops).  (5)  Blot  with  absorbent 
paper  and  dehydrate  rapidly  with  95%  and  absolute  alcohol.  (6) 
Differentiate  carefully  with  creosote,  to  the  right  degree;  (7)  blot 
with  absorbent  paper,  and  rinse  in  several  changes  of  xylene.  Mount 
in  balsam.  Neuroglia  fibers,  a  dark  blue,  neuroglia  'cells'  a  light 
blue,  axis  cylinder  and  myelinic  sheath  red  to  brown,  nuclei  dark  blue, 
etc. 

This  method  may  be  used  with  fresh  or  formalin  material,  human 
or  animal.  (Kingery). 

THE    BLOOD. 

Special  methods  in  the  examination  of  the  blood  include  (1) 
Examining  fresh;  (2)  Technic  of  staining  blood  films;  (3)  Deter- 
mination of  the  number  of  red  and  white  corpuscles  per  cubic  milli- 
meter; (4)  differential  counting  of  the  white  corpuscles;  (5)  De- 
termination of  the  relative  amount  of  hemoglobin;  (6)  Spectroscopic 
examination  of  blood  (hemoglobin),  etc.  (1)  and  (2)  are  briefly 
given  here;  for  (6)  see  [17]. 


80 

§  210.  Examining  fresh.  This  consists  in  covering  a  drop 
on  a  slide,  and  immediately  sealing  the  cover-glass  to  prevent  evapo- 
ration, observing  the  following  cautions:  (1)  The  drop  of  blood 
(from  the  finger  or  the  lobe  of  the  ear)  should  flow  freely  and  not 
be  obtained  by  pressure.  The  drop  should  be  a  medium-sized  one, 
which  will  spread  out  in  an  even,  thin  layer  under  the  cover.  (2) 
The  drop  should  be  received  upon  a  cover  or  slide,  covered,  and 
sealed  at  once  with  castor-oil. 

Examination  of  fresh  blood  may  be  used  in  clinical  examination 
for  the  detection  of  some  abnormal  conditions,  and  it  is  of  value  in 
the  rough  diagnosis  of  many  others. 

§  211.  Stained  preparation  of  blood,  (a)  Preparing  the  blood 
film.  This  may  be  best  done  in  one  of  two  ways:  (1)  The  edge 
of  a  slide  is  first  drawn  through  a  drop  of  fresh  blood  and  then  moved 
quickly  across  the  surface  of  a  clean  cover-glass  or  slide,  in  this 
way  spreading  the  blood  in  a  thin,  even  layer  upon  the  glass.  Success 
depends  upon  getting  the  right  amount  of  blood  upon  the  edge  of  the 
slide  and  the  quick,  even  movement  by  which  it  is  spread  upon  the 
cover-glass  or  slide.  Preparing  the  film  on  a  slide  is  simpler  and  to  be 
preferred  if  a  differential  count  of  the  leucocytes  is  to  be  made.  A 
second,  possibly  better,  method  is  the  following: 

(2)  Have  ready  twro  thin  clear  cover-glasses  (or  slides)  and 
obtain  a  drop  of  fresh  blood.  Take  one  of  the  covers  in  the  forceps, 
touch  it  to  the  drop  of  blood  and  place  it  upon  the  second  cover- 
glass  eccentrically,  with  one  edge  projecting  slightly.  Slip  the  two 
covers  apart  in  the  plane  of  their  surfaces  and  dry  them  quickly  by 
waving  them  in  the  air  or  by  passing  them  rapidly  over  the  tip  of 
a  flame.  The  lower  cover-glass  will  have  the  better  film. 

(b)  Fixing  the  hemoglobin  with  (a)  ether-alcohol  or  heat,  or  (b) 
at  the  time  of  staining  (methyl  alcohol)  (§  214). 

§  212.  Fixing  the  film.  When  the  blood  films  on  the  covers 
are  dry,  place  them  in  the  fluid  for  J/2  to  1  or  several  hours.  Let 
them  fix  for  a  longer  rather  than  a  shorter  time,  as  the  quality  of 
the  stain  (with  triacid  mixture)  will  be  improved.  After  they  have 
fixed  a  sufficient  time  remove  and  again  dry  them  in  the  air.  They 
may  now  be  stained,  immediately  or  at  convenience. 

§  213.  Staining  unfixed  films.  Eosin-Methylene  Blue  stains 
(below).  Fixed  films  may  be  stained  writh  hematoxylin  and  eosin  as 
well  as  with  other  stains.  If  the  film  is  on  the  slide  balsam  and  a 


81 

cover-glass  are  unnecessary  if  it  is  to  be  examined  with  the  oil  immer- 
sion objective. 

§  214.  Eosin-Methylene  Blue.  The  most  of  the  formulas  are 
made  on  the  principle  of  neutral  stains, — eosinates  of  methylene  blue 
dissolved  in  methyl  alcohol,  the  staining  solution  being  diluted  with 
water  during  the  staining  (§  85,  b).  As  polychrome  methylene  blue 
is  generally  used,  the  range  of  selectivity  is  increased  by  the  presence 
of  methylene  azure. 

(a)  Nochts-Hastings   Stain.     (§    130).      Stain    blood    smears    1 
minute  with  the  undiluted  stain,  then  dilute  with  distilled  water 
until  a  metallic  film  begins  to  appear  and  the  diluted  stain  appears 
reddish  at  the  edge.     Stain  5  minutes  more,  rinse  quickly  with  dis- 
tilled water,  absorb  excess  with  absorbent  paper,  dry  in   the   air; 
when  dry  mount  in  balsam. 

(b)  Wright's  stain   (§   129)   is  of  similar  composition  and  the 
staining  process  is  carried  out  in  the  same  manner.     Stain  blood 
smears  1  minute  with  the  undiluted  stain ;  dilute  drop  by  drop  with  an 
equal  volume  of  water  and  stain  for  3  minutes;    rinse,  dry,  etc.,  as 
above. 

(c)  Jenner's  stain  (§  128)  is  a  simpler  stain,  and  easier  to  use. 
The  simple   (i.  e.,  not  polychrome)   methylene  blue  is  used,  and 
the  differentiation  of  the  stain  is  secured  in  the  washing  out.     Stain 
blood  films  3  minutes  or  more,  rinse  a  short  time  with  distilled  water 
(until  the  best  portions  of  the  film  are  pink) .     Absorb  excess  of  water 
with  absorbent  paper  and  dry  in  the  air.     When  dry,  mount  in 
balsam. 

§  215.  It  is  sometimes  advantageous, — as  for  class  work  and  with 
non-mammalian  blood, — to  handle  blood  in  bulk.  The  following 
method  has  been  used  with  good  results.  Fix  for  1  to  6  hours  by 
having  the  blood  drop  into  a  vial  of  1%  osmic  acid.  The  blood-cells 
are  allowed  to  settle  and  the  supernatant  fluid  removed  with  a  pipette. 
By  this  method  the  blood  is  passed  through,  successively, — 2  or  3 
changes  of  distilled  water,  50%,  67%  alcohols,  paracarmine,  67%, 
82%,  95%  and  absolute  alcohols,  xylene,  to  thin  xylene  balsam  (§  161) 
in  which  the  blood  is  stored.  By  gently  agitating,  the  corpuscles  are 
evenly  distributed  and  a  drop  of  the  balsam  mounted  contains 
numerous  blood  cells.  This  is  an  excellent  method  for  preparing 
isolated  epithelial  and  muscle  cells  for  class  use.  Such  material  may 
be  kept  for  years  and  is  always  ready  to  use. 


82 

FINE    INJECTION. 

For  the  purpose  of  examining  microscopically  the  finer  arteries 
and  veins  and  the  capillaries  in  a  tissue,  and  their  relation  to  the 
other  parts,  it  is  necessary  to  fill  them  with  some  colored  injection 
mass,  or  otherwise  stain  or  color  them.  Numerous  injection  masses 
are  in  use;  the  following  meet  the  general  needs.  For  injection 
fluids  for  special  purposes,  consult  the  literature  [6]. 

§  216.  Carmine  gelatin  mass.  Formula:  Dry  gelatin,  75 
grams;  carmine  (No.  40),  10  grams;  water,  90  c.  c.;  ammonia,  10 
c.  c.;  acetic  acid,  q.  s.\  chloral  hydrate,  10  grams. 

Soak  the  gelatin  in  water  until  it  is  soft;  pour  off  the  superflu- 
ous water  and  melt  it  (in  an  agate  or  porcelain  dish)  over  a  water 
bath.  Grind  the  carmine  to  a  paste  with  water;  add  all  the  am- 
monia and  water;  filter,  warm  to  80°  or  90°  C.,  and  add  to  the  warm 
gelatin.  Then  add  slowly  the  acetic  acid  diluted  with  an  equal  vol- 
ume of  water,  while  constantly  stirring  the  mass,  until  the  mass 
smells  very  slightly  of  the  acid.  Filter  through  fine  flannel.  If  the 
mass  is  acid,  the  chloral  hydrate  may  be  safely  added  (as  a  preserva- 
tive) ;  if  any  ammonia  is  present  it  will  decompose  it  forming  chloro- 
form, and  a  granular  precipitate.  If  too  much  acid  is  added,  the 
gelatin  will  not  set. 

§  217.  Berlin  blue  injection  mass.  Formula:  Dry  gelatin, 
75  grams;  saturated  aqueous  solution  of  Berlin  blue,  150  c.  c.;  chloral 
hydrate,  10  grams.  Prepare  the  gelatin  in  the  manner  given  above 
(§  216);  warm  the  Berlin  blue  solution  (to  80°  or  90°  C.),  and  add 
it  to  the  hot  gelatin.  Heat  the  mixture  for  10  minutes  or  more, 
stirring  it  occasionally,  and  filter  it  through  fine  flannel  and  add  the 
chloral  hydrate. 

§  218.  For  securing  the  best  results  in  injecting  the  following 
conditions  should  be  observed:  (1)  A  young  but  nearly  mature, 
lean  animal  is  to  be  preferred.  (2)  Kill  the  animal  with  an  anes- 
thetic (chloroform)  and  leave  it  in  the  anesthetic  at  least  half  an 
hour  before  beginning  the  injection;  do  not,  however,  wait  until 
rigor  mortis  sets  in.  (3)  Inject  only  the  part  desired,  tying  all  an- 
astomosing vessels  and  all  vessels  to  other  parts.  Inject  into  the 
artery  of  the  part,  leaving  the  vein  open  until  nearly  pure  injection 
mass  escapes,  then  tie  it  and  continue  the  injection  until  the  part 
feels  hard  and  is  the  color  of  the  injection  mass.  (4)  When  the 
injection  is  finished  cool  the  part  injected  by  means  of  cold  water, 
ice,  or  snow. 


83 

(5)  Harden  the  injected  tissue  1  or  2  days  in  50%  alcohol,  2 
or  3  days  in  67%  and  82%  alcohols.  The  acidity  of  the  alcohols 
should  be  insured  by  adding  to  the  50%  alcohol  a  few  drops  of  acetic 
acid.  The  tissue  may  be  stored  in  82%  alcohol  until  ready  for  sec- 
tioning. Formalin  (10%)  may  also  be  used  as  the  fixer  and  preserving 
fluid.  For  sectioning  the  celloidin  method  is  usually  preferable. 

§  219.  Silvering  blood  vessels.  Silver  nitrate  may  be  used 
for  coloring  blood  vessels,  and  thus  differentiating  them.  See  §  222. 

§  220.  Dense  Masses,  such  as  do  not  pass  through  the  capil- 
lary network  are  useful  in  giving  double  injections,  the  veins  and 
capillaries  one  color,  arteries  another.  Two  such  may  be  mentioned: 
(a)  Lampblack  Gelatin  Mass;  (b)  Ultramarine  Gelatin  Mass  (Spal- 
teholz.). 

(a).  Lampblack  and  gelatin  mass  in  the  proportion  of  about 
1:12. 

(b).  A  10%  gelatin  mass  to  which  is  added  ultramarine  in 
proportion  of  30:100. 

In  injecting,  inject  through  the  artery  with  one  of  the  gelatin 
masses  given  in  §  §  216 — ,  and  follow  it  up  with  one  of  the  above 
which  will  push  the  first  mass  through  into  the  capillaries  and  veins. 


SILVER    NITRATE    IMPREGNATIONS. 

§  221.  The  preparations  stained  by  means  of  nitrate  of  silver 
are  prepared  as  follows;  The  fresh  tissue  is  washed  for  a  minute  or 
so  in  distilled  water  to  remove  from  the  surface  all  albuminous  sub- 
stance, and  then  transferred  for  2  to  5  minutes  or  longer  to  a  1  to 
Yf/o  aqueous  solution  of  silver  nitrate  and  exposed  to  direct  sunlight 
until  a  light  brown.  When,  by  examination  with  the  microscope,  the 
stain  was  found  to  be  sufficient  it  was  again  rinsed  in  water  and  placed 
in  glycerin  or  alcohol.  Employed  in  this  manner  with  fresh  tissue, 
silver  nitrate  stains  the  cell  cement,  affording  thus  negative  images  of 
the  cells.  If  a  membrane  such  as  mesentery  is  to  be  silvered  removed 
from  the  body,  it  should  first  be  cautiously  stretched,  as  over  a  ring, 
to  avoid  creases. 

§  222.  Silvering  Vascular  Epithelium.  In  order  that  the 
vascular  epithelium  of  small  arteries,  veins,  and  capillaries  should  be 
well  demonstrated,  silver  nitrate  solutions  of  J^  to  //2%  strength 
must  be  injected  into  the  vessels. 


84 

§  223.  Procedure.  Connect  a  canula  with  the  artery  supply- 
ing the  alimentary  canal  (superior  mesenteric)  or  the  brain  (caro- 
tid) and  inject  distilled  water  until  the  water  flows  out  of  the  re- 
turning vein  colorless.  Then  immediately  inject  the  silver  solution 
until  it  runs  from  the  vein.  After  a  minute  or  two  follow  writh 
distilled  water  or  physiological  salt  solution.  Place  the  intestines 
and  mesentery  in  water  and  expose  them  to  the  light  until  they  become 
slightly  browned.  Strips  of  the  muscular  coat  of  the  intestines, 
especially  of  the  rabbit,  will  show  capillaries  well.  Veins  and  arteries 
side  by  side  may  be  found  in  the  mesentery.  If  the  brain  vessels  are 
injected  one  can  get  admirable  preparations  showing  nuclei  as  well  as 
cell  outline  by  staining  in  hematoxylin.  Mount  in  glycerin,  or,  if 
desired,  dehydrate  and  mount  in  balsam.  The  tissue  may  be  kept  in 
50%  alcohol  or  in  50%  glycerin  for  several  months  before  mounting 
if  it  is  kept  in  the  dark. 

For  large  vessels  and  endocardial  epithelium  open  the  vessels 
or  the  heart  and  silver  as  directed  above  for  mesentery.  It  may  be 
necessary  to  make  thin  free-hand  sections  so  that  the  preparation 
will  be  thin  enough  for  high  powers. 

HISTO-CHEMICAL   METHODS. 

There  are  special  chemical  substances  which  it  is  often  desirable 
to  preserve  and  differentially  stain.  In  most  cases,  the  staining  re- 
actions are  not  specific  enough  to  come  under  the  category  of  micro- 
chemical  tests,  the  evidence  gained  being  circumstantial  or  indirect, 
the  application  of  two  or  three  different  methods  being  sometimes 
necessary  for  confirmation.  Such  methods  may  be  spoken  of  as 
histo-chemical  rather  than  micro-chemical. 

A.    Fats.     (Lipoids). 

§  224.  Free  fats  and  lipoids  are  soluble  in  ether,  chloroform, 
absolute  alcohol,  xylene,  benzene,  and  essential  oils.  As  these  are 
necessary  for  paraffin  and  celloidin  imbedding  methods,  especially 
the  former,  the  satisfactory  preservation  of  these  substances  pre- 
sents some  difficulties.  The  use  of  the  freezing  microtome  is  there- 
fore particularly  called  for.  See,  however,  §  §  227,  228. 

The  stains  applicable  to  the  demonstration  of  fats  are  (a)  stains 
soluble  in  the  fat  solvents,  e.  g.,  Sudan  III,  and  scarlet  red;  and  (b) 
such  as  depend  upon  the  reduction  of  salts  by  the  fats  (e.  g.,  osmic 
acid  and  potassium  dichromate)  [29]. 


85 

§  225.  Sudan  III.  Fix  tissue  in  formalin  or  Miiller's  fluid. 
Cut  free-hand  sections,  employ  the  freezing  microtome  method,  or 
isolation  (§  §  35 — ).  Rinse  sections  in  82%  alcohol  and  transfer 
sections  to  a  strong  solution  of  the  stain  in  82%  alcohol; 
leave  several  minutes  covered  from  evaporation;  rinse  with  82% 
alcohol  and  transfer  to  water.  Mount  in  glycerin  or  glycerin  jelly 
(§  §  151,  155).  Fat  is  stained  red. 

§  226.  Herxheimer's  Stain.  Preparation  for  staining  is  as  above 
(§  225).  Pass  sections  into  67%  alcohol.  Transfer  to  a  strong  solu- 
tion of  Scarlet  Red  in  67%  alcohol  rendered  alkaline  by  2%  of  sodium 
hydroxid.  Stain  for  several  minutes;  rinse  with  67%  alcohol  and 
transfer  to  water.  Mount  in  glycerin  or  glycerin-jelly.  Fat  globules 
stained  red.  It  affords  a  more  intense  stain  than  Sudan  III. 

Indophenol  (saturated  solution  in  67  or  82%  alcohol)  may  be 
used  as  a  blue  stain  for  fat  in  a  similar  manner. 

§  227.  Osmic  Acid.  (§  §  30,  18,  19).  Osmic  acid  is  reduced 
by  the  unsaturated  fatty  acids  (e.  g.,  oleic  acid)  which  are  blackened 
by  it.  The  saturated  fats  and  fatty  acids  (e.  g.,  stearic,  palmitic,) 
are  not  so  blackened  [1]  but  the  black  color  subsequently  appears 
when  the  tissue  is  placed  in  alcohol  [41].  The  fat  so  oxidized  and 
impregnated  with  osmium  (?)  becomes  less  soluble  in  fat  solvents 
(§  224)  and  may  be  retained  in  tissue  imbedded  in  either  paraffin  or 
celloidin.  There  is,  however,  a  difference  in  fats;  adipose  tissue  is 
easily  preserved,  while  some  of  the  fat  granules  found  in  the  organs 
require  the  special  precautions  mentioned  below. 

Fix  sections  of  tissue  2  to  3  mm.  thick  in  Flemming's  or  Benda's 
fluid  for  2  days;  dehydrate  in  95%  alcohol,  and  transfer  to  thin 
celloidin, — and  subsequently  thick  celloidin — made  up  with  95% 
alcohol  (not  absolute).  The  sections  may  be  stained  in  safranin, 
quickly  dehydrated  and  cleared  with  carbol-xylene  and  mounted  in 
balsam,  either  without  a  cover-glass,  or  in  thick  balsam  melted  by  heat 
and  applied  warm.  Unless  such  precautions  are  taken,  the  solvent  of 
the  balsam  may  in  time  dissolve  out  the  granules  of  blackened  fat. 

Paraffin  does  not  afford  as  good  a  preservation  of  the  more  labile 
fat  globules.  Dehydrate  in  equal  parts  of  95%  and  absolute  alcohol, 
clear  before  the  infiltration  in  carbol-xylene  or  chloroform.  Paraffin 
in  the  sections  should  be  dissolved  out  by  carbol-xylene  in  preference 
to  xylene  (§  143). 


86 

§  228.  Bichromate  mordantage,  with  subsequent  copper  or 
iron  hematoxylin  stain,  appears  to  rest  upon  the  power  of  the  fat  or 
lipoid  to  reduce  the  dichromate  and  thus  take  on  a  mordantage  which 
gives  the  basis  for  the  subsequent  staining.  So  far,  it  has  not  been 
possible  by  this  technique  to  preserve,  in  paraffin  or  celloidin  sec- 
tions, the  individual  fat  granules,  but  it  is  nevertheless  a  useful 
method  for  the  differentiation  of  lipoid-containing  cells. 

Fix  tissue  2  days  in  Zenker's  with  but  1  /10  or  1  /5%  of  acetic  acid 
or  Helly's  fluid,  mordant  4  days  or  longer  in  Muller's  fluid  at  35  to 
38°  C.,  wash  in  water,  imbed  in  paraffin  using  chloroform  as  the  clearer 
(§54).  Stain  sections  with  the  copper  hematoxylin  (§  95).  Lipoid- 
containing  cells,  myelinic  nerve  fibers,  erythrocytes,  etc.,  a  dark  blue. 
It  should  be  also  remembered  that  other  structures  may  also  be 
stained  by  this  technique. 

The  freezing  microtome  may  be  used  with  such  tissue  and  stain, 
as  has  been  done  by  Benda  and  Fischler  [6]. 

B.     Glycogen. 

Glycogen  is  soluble  in  aqueous  media,  and  while  it  may  be  retained 
by  a  short  fixation  in  several  fluids,  the  best  preservative  of  it  is  95% 
alcohol  (82%— absolute). 

§  229.  The  Iodine  Method.  [16].  Fix  tissue  in  95%  or  82% 
alcohol.  Imbed  in  paraffin.  Spread  the  sections  using  instead  of  the 
water,  the  iodine  stain  for  glycogen,  which  is  made  up  as  follows: 
iodin,  \^/2  grams;  potassium  iodid,  3  grams;  sodium  chlorid,  \}/<i 
grams;  distilled  water,  300  c.  c.  Spread  sections  may  be  stained  or 
restained  by  immersing  in  the  iodin  solution  which  will  color  the 
glycogen  a  mahogany  red.  For  very  soluble  glycogen,  50%  alcohol 
may  be  employed  instead  of  the  water  in  making  up  the  stain.  In 
mounting,  dissolve  the  paraffin  with  xylene,  drain,  place  on  the 
preparation  melted  yellow  vaseline,  cover,  seal  with  shellac  or  balsam. 

§  230.  Best's  Method.  [5].  While  rather  complicated,  this 
is  generally  recognized  as  the  best  method  for  the  demonstration  of 
small  quantities  of  glycogen,  especially  when  it  is  desired  to  see  the 
relation  of  the  granules  to  the  protoplasm. 

Fix  tissue  in  95%  alcohol;  imbed  (preferably)  in  celloidin  (§67); 
if  paraffin  is  used,  after  dehydration  (§  51)  place  the  pieces  of  tissue 
in  pure  acetone  for  15  minutes,  xylene  20  minutes,  xylene  paraffin 
1  hour,  pure  paraffin  (§  52)  1  hour.  Stain  sections  in  Delafield's 
hematoxylin  (§  91)  strongly,  rinse  and  differentiate  (if  necessary) 
and  stain  in  the  following  special  carmine  stain  which  had  been 


'  87 

previously  prepared,  (a).  Carmine,  1  gram;  ammonium  chlorid, 
2  grams;  lithium  carbonate,  0.5  grams;  distilled  water,  50  c.  c. 
Bring  the  mixture  to  a  boil.  When  it  is  cool,  add  20  c.  c.  10%  am- 
monia. Preserve  the  solution  in  the  dark;  after  2  to  3  days  it  is 
ready  for  use  and  retains  its  staining  quality  for  a  few  weeks  only, 
(b)  When  ready  to  stain,  filter  the  above  solution  (a),  and  add  to 
2  parts  of  the  stain,  3  parts  of  10%  ammonia  solution,  and  6  parts 
of  methyl  alcohol. 

Stain  sections  1  hour,  differentiate  in  a  mixture  of  2  parts  of 
methyl  alcohol,  4,  parts  of  absolute  alcohol,  5  parts  of  water;  rinse 
with  82%  alcohol,  dehydrate,  clear  and  mount  in  balsam.  Glycogen 
stained  an  intense  red. 

In  working  with  glycogen  it  is  sometimes  necessary  to  apply  as 
a  control  the  digestion  of  the  glycogen  in  one  or  more  sections  or  a 
part  thereof,  by  means  of  saliva. 

C.  Amyloid. 

Amyloid,  a  form  of  connective  tissue  degeneration,  resembles 
glycogen  in  some  of  its  physical  (not  chemical)  properties  and  stain- 
ing reactions.  Two  methods  for  its  demonstration  may  be  men- 
tioned : 

§  231.  Iodine  Method.  Practically  any  fixer  may  be  used 
(Zenker's  fluid).  Paraffin,  celloidin,  or  (better)  frozen  sections 
may  be  used.  Stain  sections  with  the  iodine  solution  (§  229)  for 
several  minutes;  rinse  in  distilled  water  and  transfer  to  glycerin  or 
glycerin-jelly  (§  151,  155)  in  which  they  may  be  mounted.  Seal  the 
preparations  (§  162 — ).  The  amyloid  a  reddish-brown;  the  stain, 
however,  will  fade  in  the  course  of  a  few  months. 

§  232.  Gentian  Violet,  among  other  anilin  stains,  colors  amyloid 
differentially  (metachromasia).  Stain  paraffin  sections  for  several 
minutes  in  a  1%  solution  of  the  stain;  rinse  and  differentiate  in  1% 
acetic  acid;  wash  thoroughly  with  distilled  water,  and  (a)  mount  in 
glycerin- jelly,  or  (b)  dry  in  the  air,  treat  with  xylene  and  mount  in 
balsam.  In  the  latter  procedure,  the  staining  may  be  applied  before 
removing  the  paraffin. 

D.  Mucus. 

Mucous  substances  (mucins,  mucinoids)  possess  acid  properties 
combining  with  alkalis  and  bases  (heavy  metals),  such  combinations 
swelling  up  or  dissolving  in  water.  Acetic  acid,  alcohol  and  picric 
acid  also  precipitate  these  substances.  For  the  fixation  of  mucus, 


88 

most  fixers  may  be  used, — Mercuric  chlorid,  Zenker's  fluid,  Flem- 
ming's  fluid  or  Hermann's  fluid.  If,  however,  it  is  desired  to  pre- 
serve the  mucus  granules  (mucinogen?)  in  the  cell-body,  alcoholic 
or  picric  acid  fixers  are  usually  required,  and  the  tissue  at  no  time 
subsequently  should  be  placed  in  water  or  aqueous  solutions.  Be- 
cause of  its  acid  character  mucus  stains  wTith  basic  stains,  the  first 
two  methods  given  below  being  very  selective.  If  cell  granules  are 
desired,  remember  the  caution  as  to  the  avoidance  of  water. 

§  233.  Mucicarmine.  (§  103).  Fix  as  recommended  above. 
Stain  paraffin  sections  1  to  24  hours;  wash  in  water,  dehydrate, 
clear  and  mount  in  balsam.  It  may  be  used  diluted  with  50%  or 
67%  alcohol. 

§  234.  Muchematein.  (§  93).  Employ  in  the  same  manner  as 
mucicarmine.  The  alcoholic  formula  is  indicated  if  cell  granules 
are  to  be  preserved. 

Staining  of  the  nuclei  with  hematoxylin  (§  88,  89)  or  carmine  (§  98) 
or  elastin  stain  (§  125)  may  be  previously  given;  a  picro-fuchsin 
stain  may  follow  it.  Excess  of  alum  (aluminium  salts)  or  presence 
of  acid  must  be  avoided,  hence  wash  thoroughly  with  water  if  there 
has  been  previous  staining. 

§  235.  Basic  anilin  stains  usually  give  sharp,  often  differen- 
tial stains  (metachromasia)  for  mucus.  Methylene  blue,  toluidin 
blue,  or  gentian  violet  may  be  recommended.  Safranin  after  Flem- 
ming's  fluid  fixation  often  gives  a  delicate  stain  for  mucus. 

E.     Micro-chemical  Tests. 

Micro-chemical  tests  for  inorganic  elements  must  occasionally 
be  employed,  particularly  in  cytological  work.  The  methods  have 
been  largely  elaborated  by  Macallum  to  whose  papers  references  are 
given. 

§  236.  Iron.  (a).  Berlin  Blue  Reaction.  Alcohol  fixation  and 
the  freezing  microtome  should  be  employed.  Rinse  sections  in 
distilled  water,  2%  potassium  ferrocyanate,  4  to  5  hours,  1%  Hcl 
alcohol  several  hours,  rinse  in  water,  dehydrate,  clear  with  clove  oil; 
balsam.  A  carmine  stain  may  be  given, — before  or  afterwards. 

(b)  Ammonium  Sulphide  Reaction.  Place  sections  in  am- 
monium sulphide  solution  (freshly  prepared)  for  5  to  20  minutes; 
(dark  green  color);  rinse  quickly  with  water,  dehydrate,  clear  in 
clove  oil,  mount  in  balsam. 


89 

Free  iron  may  also  be  demonstrated  by  the  use  of  a  pure  aque- 
ous solution  of  hematoxylin  (dark-blue  color);  other  metals,  how- 
ever, also  unite  with  hematoxylin  (e.  g.,  calcium). 

§  237.  Masked  Iron  does  not  give  the  reaction  with  pure  aqueous 
hematoxylin  [31,  34].  Macallum  applies  the  ammonium  sulphide  at 
higher  temperature  (60°  C.)  and  for  several  days. 

§  238.     Phosphorus.     See  Macallums  methods  [31]. 

§  239.     Potassium.     See  Macallum's  method  [33]. 

§  240.     Chlorides.     See  Macallum's  method  [32].     Cautions! 

§  241.  Calcium  stains  strongly  with  aqueous  hematoxylin  and 
may  often  be  so  demonstrated.  To  identify  the  calcium  and  dis- 
tinguish from  iron,  usual  chemical  tests  may  be  applied  [31,  28]. 


90 

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25.  Heidenhain,   M.     Ueber  Vanadiumhaematoxylin,    Picro-blauschwarz  und 

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26.  Heidenhain,    M.     Plasma   und    Zelle.     Erste   Abt.:     Allg.    Anatomie    des 

lebendigen  Masses.     Jena.  1907. 

27.  Jenner,  A.,    A  new  Preparation  for  Rapidly  Fixing  and  Staining  Blood. 

Lancet,  1899, 1.     p.'370. 

28.  v.     Kahlden-Giercke.     Technik     der     histolgischen     Untersuchungen.     8. 

Ed.  Fischer,  Jena,  1909. 

29.  Faure-Fremiet,  A.     Mayer,  G.  Schaeffer.     Sur  la  microchimie  des  corps 

gras;     application   a   1'etude   des   mitochon dries.     Arch.     d'Anat.    mi- 
croscopique.     Vol.  XII,  Fasc.  L,  1910.     pp.  19-102. 

30.  Lee,   A.    B.     The   Microtomist's   Vademecum.     7   Ed.     Blakiston,    Phila. 

1913. 

31.  Macallum,  A.  B.     Die  Methode  und  Ergebnisse  der  Microchemie  in  die 

biologische"  Forschung.     Ergeb.    d.    Physiol.     Vol.    VII,    pp.   552-652. 
1908. 

32.  Macallum,  A.  B.     On  the  Nature  of  the  Silver  Reaction  in  Animal  and 

Vegetable  Tissues.     Proc.  Roy.  Soc.  Vol.  LXXVI.     1905.     p.  217. 

33.  Macallum,  A.  B.     On  the  Distribution  of  Potassium  in  Animal  and  Vegeta- 

ble Cells.     Journ.  Physiol.     Vol.  XXXII.     p.  95.     1905. 


92 

34.  Macallum,  A.  B.     On  a  new  Method  of   distinguishing  between  organic 

and  inorganic  Iron.  Journ.  Physiol.  Vol.  XXII.  1897.  p.  92.  Also: 
On  the  Demonstration  of  the  Presence  of  Iron  in  Chromatin  by  Micro- 
chemical  Methods.  Proc.  Roy.  Soc.  Vol.  L,  p.  277.  1891. 

35.  Mallory    &    Wright.     Pathological    Technique.     5th.    Ed.    1911.     W.    B. 

Saunders  &  Co.,  Philadelphia  and  London. 

36.  Mann,  Gustav.     Physiological  Histology.     Oxford.     1903. 

37.  Moore,  V.  A.     A  Note  on  the  Use  of  Anise  Oil  in  Histological  Methods 

with  special  Reference  to  its  Value  in  Cutting  Serial  Sections  on  the 
Freezing  Microtome.  Amer.  Month.  Micr.  Journ.  Vol.  XV.  1894. 
p.  373. 

38.  Myers,  B.  D.     The  Chiasma  of  the  Toad  (Bufo  lent.)  and  of  some  other 

Vertebrates.  Zeitschr.  f.  Morphol.  &  Anthropol.  Vol.  III.  1900. 
pp.  183-207. 

29.     Pappenheim,  M.     Grundriss  mikroskopischer  Farbetechnik.     Berlin.    1901. 

40.  Sheldon,  R.  E.     Paraffin-Weigert  methods  for  the  staining  of  nervous  tis- 

sue, with  some  new  modifications.  Folia  Neuro-biologica.  Vol.  VIII. 
No.  1,  1914.  pp.  1-28. 

41.  Starke,   J.     Ueber  Fettgranula  und  eine  neue  Eigenschaft   des  Osmium 

tetraoxydes.     Arch.  f.  Anat.  u.  Physiol.     Physiol.     Abt.  1895.     p.  70. 

42.  Streeter,  G.  L.     Ueber  die  Verwendung  der  Paraffineinbettung  bei  Mark- 

scheidenfarbung.     Arch.  f.  mikr.    Anat.     Bd.  LXII.     1903.     p.  734. 

43.  Strong,  O.  S.     Notes  on  the  Technique  of  Weigert's  Method  for  Staining 

Medullated  Nerve  Fibers.  Journ.  Comp.  Neurol.  Vol.  XIII.  1903. 
p.  291.  Also:  Ibid.  Vol.  XVI.  1906. 

44.  Verhoeff,  F.  H.     An  improved  differential  Elastic  Tissue  Stain.     Journ. 

Amer.  Med.  Ass'n.     Vol.  LVI,  No.  18,  pp.  1326-1327.     1911. 

45.  Wright,   J.   H.     A  rapid  Method  for  the  differential  Staining  of  Blood 

Films  and  Malarial  Parasites.  Journ.  Med.  Research.  Vol.  VII. 
1902.  p.  138. 

46.  Wright,  J.  H.     Revised  Directions  for  making  and  using  the  Wright's  blood- 

stain.    Journ.  Amer.  Med.  Ass'n.     Vol.  LV.     pp.  1979.     Dec.  3,  1910. 


INDEX 


Acetic  acid,  picric,  formol,  12 
Acetic  acid,  5,  7 

Acid  hematoxylin  (Delafield's),  39 
Acid,  formic,  15,  69 

fuchsin,  44 

hydrochloric,  36,  63 

nitric,  15,  1 8,  63,  66 

osmic,  15,  18,69,73,75,85 

sulphuric,  18 
Acid  stains,  35 
Acid  violet,  45 
Action  of  fixer,  7,  8 
Adjective  staining,  35 
Albumin  fixative,  49 
Alcohol,    absolute,    95%,    82%,    67%, 

50%,  ii,  14,22,50,51 
Alcohol-acetic,  14 
Alcohol-acetp-chloroform,  14 
Alcohol-acetic-formol,  14 
Alcohols  as  fixers,  14,  60,  70,  71 
Alcohol,  dehydration  in,  22,  27,  51,  54 
Alcohol,  grades,  preparation  of,  1 1 
Alum  cochineal,  41 
Alum  differentiator,  36,  70 
AlkaUne  methylene  blue,  42 
Ammonium  molybdate,  78 
Ammonium  pi  crate,  78 
Ammonium  sulphid  (iron  test) ,  88 
Amyloid,  stains  for,  87 
Anilin  blue,  Mallory's  connective  tissue 

s£ain,  44 

Anise-seed  oil,  33 
Babes'  safranih,  42 
Balsam,  Canada,  54,  55 
Balsam,  camsal,  55 

Damar,  52 

Euparal,  55 
Basic  stains,  35,  88 
Benda's  fluid;   see  Flemmings  fluid 
Benda's  stain  for  neuroglia,  79 
Bensley's  neutral  gentian,  46 
Berlin  blue,  82 

Berlin  blue  reaction  (iron),  88 
Best's  method  for  glycogen,  86 
Bleaching;     peroxid,    perhydrol;     see 

Hermann's  fluid 
Bleu  de  Lyon,  stain,  45 
Blocks,  wooden,  for  imbedding,  28 
Blood  films,  fixing  and  staining,  80,  81 
Blood  methods  for,  79 
Blood,  preserving  in  bulk,  81 
Blood  vessels,  silvering,  83 
Bone,  63,  65 
Borax  carmine,  40 

Bouin's  fluid,  see  picro-aceto-formol 
Boxes,  paper,  for  imbedding,  24,  29 
Cajal,  methods  for  neurofibrillae,  71 


Calcium,  tests  for,  89 

Camsal  balsam,  55 

Carbol-xylene,  55 

Carmalum,  40 

Carminates,  carmine,  stains,  40,  86 

Carmine  gelatin  mass,  82 

Carminic  acid,  40 

Carnoy's  fluids,  14,  70,  79 

Castor-xylene,  29,  30 

Caustic  potash,  dissociator,  19,  67 

Cedar  wood  oil,  clearer,  23 

Cell,  methods  for,  59 

Celloidin,  staining,  of  37 

Celloidin  method,  20,  26-, 

Celloidin,  solutions,  2%,  6%,  12%,  27, 

28 

K%,34,49 

removal  of ,  48 
Chick  blastoderm,   fixing;     see  nitric 

acid 

Chloral  hematoxylin,  88 
Chlorides,  test  for,  89 
Chloroform,  clearer,  23,  69 

fixer,  14 

hardener,  29 
Chromatin,  stains  for,  59 
Chromo-aceto-osmic;    see  Flemming's 

fluid 

Chromo-nitric;  see  Perenyi's  fluid 
Clarifier,  clarifying,  29,  30 
Class,  isolated  tissue  for,  81 
Cleaning  mixture  for  glass,  58 
Cleaning    slides,    cover-glasses,    glass- 
ware, 57,  58 

Clearers,  clearing,  22,  23,  55 
Cochineal,  alum,  41 
Collagen,  staining,  61 
Collodion;   see  celloidin 
Congelation  mass;    see  freezing 
Congo  red,  43,  69 
Connective  tissue,  61 
Copper    dichromate-sublimate  -acetic, 

12,61,72,79 

Copper  hematoxylin,  40,  73,  86 
Cover-glasses',  57,  58 
Crystallization  of  paraffin  mass,  25 
Cytoplasm,  fixation,  60 
Cytoplasm,  granules,  60,  6 1 
Damar  balsam,  mounting  medium,  52 
Decalcification,  63 
Dehydration,  22,  27,  54 
Delafield's  hematoxylin,  39,  69 
Dichromate  as  fixer,  8,  1 6,  72,  73,  74,  86 
Dichromate-acetic,  12 
Differentiation,  36,  73  ' 
Digestion  method  (reticular  tissue),  62 
Dishes,  for  paraffin  imbedding,  23 


94 


Dissociation,  dissociators,  6,  16- 
Double  staining,  double  stains,  37 
Dry  mounting,  52 
Ehrlich's  acid  hematoxylin,  39 
Ehrlich-Biondi-Heidenhain  triple  stain, 

42 

Elastic  fibers  (elastin),  stains  for,  45,  62 
Eosin,  43 

Eosin-methylene  blue,  42 
Eosinate  of  methylene  blue,  46 
Epithelium,  silvering,  83 

isolation  (dissociation)  of,  17,  1 8 
Erlicki's  fluid,  15 
Erythrosin,  43 
Ether-alcohol,  27 
Euparal,  55 

Fastening  sections  to  slide,  47- 
Fat,  fixation  of,  72,  84 

freezing  microtome,  use,  84 

stains  for,  84 
Fixation,  7,  8 
Fixation  by  injection,  9 
Fixation,  rules  for,  9 
Fixers,  action  of,  7 

formulae  for,  11-16 

list  of,  8 
Fixing,  6,  7 

Flemming's  fluid,  13,  74,  85 
Flemming's  triple  stain,  43 
Formaldehyde,  formalin,  14,  15 

Dissociator,  18,  68 
Formol-dichromate,  12 
Free-  (hand)  sections,  47,  48 
Freezing  method  for  sectioning,  19,  33, 

48,84 

Fresh  tissue,  examination  of,  6 
Fuchsin  acid,  44 
Gage's  dissociators,  18 
Gage's  hematoxylin,  38 
Gage's  method  for  glycogen,  86 
Gelatin  masses,  82 
Gentian  violet,  41,  43,  46,  87,  88 
Gilson's  fluid;    see  mer euro-nitric 
Glycerin,  53,  55 
Glycerin- jelly,  54,  56 
Glycogen,  fixation,  staining,  86 
Gold  chlorid,  methods,  69 
Golgi  methods,  74 
Gram's  solution;   see  iodine,  45 
Granules,  cytoplasmic,  60 

secretion,  61 
Ground  sections,  64 
Gum  arabic,  33 
Hsemalum,  Mayer's,  39 
Hair,  dissociation  of,  18,  19 
Hardening  of  tissues,  10 

celloidin  mass,  29 
Hasting's  stain,  46,  81 
Heidenhain's  iron  hematoxylin,  39 
Held's  method  (nerve  cells) ,  70 


Kelly's  fluid,  12 
Hemateates,  hematein,  38 
Hematoxylin  stains,  formulae,  38,  39,  40 

stock  solution,  38 
Hermann's  fluid,  13 
Herxheimer's  stain,  85 
Horn,  dissociation  of,  18,  19 
Hydrochloric  acid  decalcifier,  63 
Hydrochloric  acid  carmine,  41 
Imbedding,  celloidin,  methods,  20,  26 

paraffin,  20,  22 
Impregnations,  36 
Indifferent  fluids,  16 
Indophenol,  85 
Infiltration,  (imbedding  methods),  22, 

26,  33 

Injection,  fine,  dense,  masses,  82,  83 
In  toto  staining,  35,  41 ,  50 
Intra  vitam  methylene  blue,  76 
lodin,  ii,  45,  86,  87 
Iron  hematoxylin,  Heidenhain's,  39 
Iron,  tests  for,  88,  89 
Isolation  ,16-,  66,  68,  69 
Jenner's  stain,  46,  8 1 
Labeling  slides,  56 
Lampblack,  gelatin  mass,  83 
Light  green,  45 
Lipoids ;   see  fats 
Lipoids;  foot  note,  7 

demonstration,  84 
Lugol's  solution;   see  iodine,  45 
Lyon's  blue,  stain;    see  Bleu  de  Lyon, 

45 

Maceration,  16 

Mallory's  connective  tissue  stain,  44 
Marchi  methods    (degenerating  nerve 

fibers),  73 

Mayer:  see  carmalum,  haemalum 
mucicarmine,  Muchematein,  par- 
acarmine 

Mercuric  chlorid,  1 1 
Mercuro-nitric,  13 
Methylene  blue,  42,  70,  76 
Methyl  green,  42 
Micro-chemical  tests,  58 
Microtome  knife,  care  of,  58 
Mitochondria,  fixation  and  staining,  60, 

61 

Mordantage,  35 
Mounting  media,  methods,  6,  52- 

cells,  53,  54 
Muchematein,  39,  88 
Mucicarmine,  41,  88 
Mucus,  87 
Miiller's  fluid,  15 

dissociator,  17 
Muscle,  isolation  of,  66 
Muscle,  methods  for,  66- 
Myelin,  myelinic  nerve  fibers,  69 
Nail,  dissociation  of,  18,  19 


95 


Nerve  cells,  isolation,  staining,  68 
Nerve    fibers,    isolation,    degenerated, 

69,73 

Nervous  system,  methods,  68- 
Neurofibrillae,  71 
Neuroglia,  stain  for,  79 
Neutral  gentian,  46 
Neutral  stains,  37,  35,  36 
Nissl's  methylene  blue,  nethod,  76 
Nitric    acid,     decalcifier,     dissociator, 

fixer,  14,  1 6,  63 
Nochts-Hastings  stain,  46,  81 
Normal,  salt  solution;  see  physiological 

salt  solution 
Orange-fuchsin  acid,  62 
Orange  G.,  44 
Orcein,  45,  62 

Orth's  fluid;  see  formol-dichromate 
Osmic  acid,  dissociator,  fixer,  15,  18,  69, 

73,  75 

Osmium  tetroxid ;  see  osmic  acid 
Pal's  method,  73 
Paracarmine,  41 

Paraffin,  grades,  method,  20,  21-,  23 
Paraffin  sections,  handling  of,  48 
Per enyi's  fluid,  14 
Peroxid,  as  bleacher;    see  Hermann's 

fluid 

Phosphorus,  89 

Physiological  salt  solutions,  16 
Picric  acid,  stain,  44 
Picro-aceto-formol,  12 
Picro-fuchsin,  44,  62 
Picro-nitric,  14 
Platino-aceto-osmic :     see     Hermann's 

fluid 

Potassium  dichromate,  fixer,  16 
Potassium  hydroxid  dissociator,  19,  67 
Potassium,  tests  for,  89 
Progressive  staining,  35 
Pyridin;   see  Cajal's  silver  methods,  71 
Pyroxylin;    see  celloidin 
Quadruple  stains,  89 
Radium,  to  prevent  electric  sections,  26 
Regressive  staining,  35 
Resorcin  fuchsin,  45,  62 
Reticular  tissue,  62 
Safranin,  42,  85 
Scarlet  red,  85 
Sealing  preparations,  55 


Secretion  granules,  61 
Sectioning,  methods  of,  6,  19- 
Section  staining,  35 
Serial  sectioning  in  celloidin,  32 
Shellac,  cells,  rings,  54 

sealing,  55,  56 
Silvering  blood  vessels,  83 
Silver  nitrate,  83,  71,  74 
Slides,  cleaning,  57 
Soluble  cotton ;  see  celloidin 
vStaining,  forms  of,  rules  of,  35 
Staining,  isolated  cells,  sections,  etc., 

18,  51,  53,  66,  67 
Stains,  35-,  38 
Stock  solutions,  1 1 
Storing  of  tissues,  10 
Substantive  staining,  35 
Sudan  III,  85 

Sulphalizarinate  of  sodium,  61 ,  79 
Sulphuric  acid  dissociator,  18 
Tellesnicky's    fluid;      see    dichromate 

acetic 

Tigroid  substance,  70 
Tissues,  fixing,  6,  7  - 

hardening,  storing,  10 
Tissue  paper,  for  handling  sections,  31 
Toluene,  as  clearer,  22,  21 
Toluidin  blue,  42 
Tooth,  63,  64 

Transferring  celloidin  sections,  31 
Triple  staining,  37 
Triple  stain;   Ehrlich-Biondi,  42 

Flemming's,  43 

Ultramarine,  gelatin  mass,  83 
van  Gehuchten's,  74 
Vascular  epithelium,  silvering,  83 
Verhceff's  elastin  stain,  45 
von   Luko's  fluid;     see   alcohol-aceto- 

formol 

Weigert  methods,  for  nerve,  71 
Weigert's  copper  hematoxylin,  40,  71 
White  connective  tissue  fibers,  61 
Wooden  blocks,   for   celloidin   imbed- 
ding, 28,  30 
Wright's  stain,  46,  81 
Xylene,  23,  50 
Xylene,  as  clearer,  22,  23 
Xylol;  see  Xylene,  22  footnote 
Zenker's  fluid,  1 1 
Zenker-formol;   see  Helly's  fluid,  12 


PRESS  OF  W.  F.  HUMPHREY,  GENEVA,  N.  Y. 


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